GARP-TGF-beta 1 antibodies

ABSTRACT

The present invention relates to antibodies and antigen binding fragments thereof, which bind to a complex of GARP and TGF-β1, particularly a complex of human GARP and human TGF-β1. These antibodies and antigen binding fragments exhibit a combination of advantageous properties including high affinity antigen binding and the ability to inhibit the release of active TGF-β from regulatory T cells. The antibodies and antigen binding fragments of the present invention are relatively resistant to deamidation, isomerization and oxidation, such that they display improved stability.

RELATED APPLICATION

This application claims benefit of priority to Great Britain ProvisionalApplication No. 1707561.5, filed on May 11, 2017, the entire contents ofwhich are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 10, 2018. Isnamed 597392_AGX5-035_ST25.txt and is 43,939 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen bindingfragments thereof, which bind to a complex of GARP and TGF-β1,particularly a complex of human GARP and human TGF-β1. These antibodiesand antigen binding fragments exhibit a combination of advantageousproperties including high affinity antigen binding and the ability toinhibit the release of active TGF-β from regulatory T cells. Theantibodies and antigen binding fragments of the present invention areimproved as compared with prior art antibodies binding to the complex ofGARP and TGF-β1. In particular, the antibodies and antigen bindingfragments of the present invention are relatively resistant todeamidation, isomerization and oxidation, such that they displayimproved stability as compared with GARP-TGF-β1 antibodies described inthe prior art.

BACKGROUND TO THE INVENTION

Regulatory T cells (otherwise known as “Tregs” or Foxp3⁺ T regulatorycells) are an important component of the immune system. In particular,Tregs play a critical role in immune homeostasis by suppressing variousaspects of the immune response. As a consequence of their role incoordinating the immune response, dysregulated Treg activity can lead tothe development of various diseases and conditions. In particular,insufficient Treg function can result in autoimmune pathology, whereasexcessive Treg activity has been linked to the inhibition of anti-tumourresponses in cancer patients.

The protein GARP (Glycoprotein A Repetitions Predominant) has beenidentified as a highly expressed marker on the surface of Tregs,particularly activated Tregs. GARP is an 80 kDa transmembrane proteinwith an extracellular region comprising 20 leucine-rich repeats. It isalso known as LRRC32. GARP serves as the receptor for TGF-β,particularly the latent form of TGF-β, and is required for theexpression of latent TGF-β on Treg cells (E M Shevach. Expert Opin TherTargets (2016) 21(2), 191-200).

TGF-β is a cytokine known to play a role in multiple processes includingcell proliferation and differentiation, tissue morphogenesis,inflammation and apoptosis. It has also been identified as an importantgrowth factor implicated in cancer development, and rather unusually,has been identified as a cytokine with tumour promoting and tumoursuppressive properties.

The production and activation of TGF-β is a multi-step process, which isregulated at different levels. TGF-β is synthesised as a pro-TGF-βdimeric precursor, each polypeptide chain consisting of alatency-associated peptide (LAP) and a mature TGF-β region. Pro-TGF-βundergoes cleavage by the enzyme furin to form “latent TGF-β,” aninactive form in which the LAP remains non-covalently associated withthe mature TGF-β region of each polypeptide chain (see FIG. 1).Membrane-localised GARP serves to transport and anchor latent TGF-β tothe cell surface of Tregs, and it is from this membrane-boundGARP-latent TGF-β complex that the active form of TGF-β is released. Avariety of mechanisms have been proposed to explain how active TGF-β isreleased from the GARP-latent TGF-β complex on the surface of Tregs.However, integrins, particularly αvβ6 and αvβ8, are now thought to playan important role in driving the shear forces needed for release of themature TGF-β dimer.

Once released, the active TGF-β dimer can act as an autocrine orparacrine mediator of downstream signalling pathways. In the context ofthe immune system, TGF-β release from Treg cells is thought to influencethe activity of various T effector cells and also Tregs themselves (seeFIG. 1). Since Tregs play an important role in suppressing immunity, itis thought that TGF-β released from Tregs and acting in an autocrinefashion may be involved in mediating Treg suppression. In particular,Treg-derived TGF-β1 is thought to play a significant role inTreg-mediated suppression of tumour immunity.

Given the role of Treg-derived TGF-β in suppressing the immune responsein the tumour microenvironment, there has been interest in targetingthis pathway as an alternative approach to cancer immunotherapy. Forexample, therapeutic agents capable of dampening this pathway may serveas useful tools to improve the efficacy of cancer vaccines or othercancer immunotherapy strategies designed to harness the power of thebody's immune system to treat cancer.

Cuende et al. (Sci Transl Med. 2015 Apr. 22; 7(284):284ra56) describesthe production and characterisation of two monoclonal antibodies (MHG-8and LHG10), which bind to the GARP-TGF-β complex on Tregs and inhibitTGF-β production. These two antibodies are also described andcharacterised in International patent applications WO2015/015003 andWO2016/125017. These antibodies were shown to be capable of inhibitingthe immunosuppressive activity of human Treg in a xenogeneicgraft-versus-host disease mouse model. This work serves to validate theGARP-TGF-β complex as a therapeutic target of interest for the purposesof modulating Treg function and consequently treating diseases such ascancer and autoimmune disease where the level of Treg activity plays animportant role. There remains a need however, for improved GARP-TGF-βantibodies capable of inhibiting TGF-β release and thereby modifyingTreg activity. The present invention addresses this problem as describedherein.

SUMMARY OF INVENTION

The present invention improves upon the state of the art by providingnew antibodies and antigen binding fragments thereof, which bind to thehuman GARP-TGF-β1 complex. The antibodies and antigen binding fragmentsof the present invention are derived from the GARP-TGF-β1 antibody“LHG-10”, described in International patent applications WO2015/015003and WO2016/125017. The heavy chain and light chain variable domainsequences of LHG-10 are shown in SEQ ID NOs: 1 and 2, respectively, andthe light chain variable domain of a chain-shuffled variant, LHG-10.6(also described in WO2015/015003 and WO2016/125017), is shown in SEQ IDNO: 3. The antibodies of the present invention differ particularly withrespect to certain CDR sequences as compared with LHG-10 and LHG-10.6,specifically with respect to the CDR2 and CDR3 sequences of the heavychain variable domain. The LHG-10 and LHG-10.6 GARP-TGF-β antibodiespossess the heavy chain CDR2 sequence: RIDPEDGGTKYAQKFQG (SEQ ID NO: 5);and the heavy chain CDR3 sequence: NEWETVVVGDLMYEYEY (SEQ ID NO: 6),whereas the antibodies of the present invention comprise the heavy chainCDR2 sequence: RIDPEDAGTKYAQKFQG (SEQ ID NO: 12); and the heavy chainCDR3 sequence: YEWETVVVGDLMYEYEY (SEQ ID NO: 13).

The differences in the heavy chain CDR2 and CDR3 sequences reportedherein result in antibodies that are improved as compared with the priorart antibodies by virtue of their improved stability. More specifically,the antibodies of the present invention are relatively resistant todeamidation, isomerization and oxidation, such that they exhibitenhanced stability. Surprisingly, these specific substitutions in theheavy chain CDR2 and CDR3 regions that lead to improved stability do notsignificantly decrease the binding affinity of the antibodies for theGARP-TGF-β1 complex. The improved stability combined with high affinitytarget binding renders the antibodies of the present inventionparticularly suitable for clinical development as therapeutic agents,for example as cancer therapeutic agents.

In a first aspect, the present invention provides an antibody or antigenbinding fragment thereof, which binds to a complex of human GARP-TGF-β1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain variable domain (VH) wherein:

the VH CDR3 comprises the amino acid sequence YEWETVVVGDLMYEYEY (SEQ IDNO: 13),

the VH CDR2 comprises the amino acid sequence RIDPEDAGTKYAQKFQG (SEQ IDNO: 12), and

the VH CDR1 comprises the amino acid sequence SYYID (SEQ ID NO: 4).

In certain embodiments, the present invention provides an antibody orantigen binding fragment thereof, which binds to a complex of humanGARP-TGF-β1, wherein the antibody or antigen binding fragment thereofcomprises a heavy chain variable domain (VH) wherein: the VH CDR3consists of the amino acid sequence YEWETVVVGDLMYEYEY (SEQ ID NO: 13),

the VH CDR2 consists of the amino acid sequence RIDPEDAGTKYAQKFQG (SEQID NO: 12), and

the VH CDR1 consists of the amino acid sequence SYYID (SEQ ID NO: 4).

The antibody or antigen binding fragment may additionally comprise alight chain variable domain (VL) wherein:

the VL CDR3 comprises the amino acid sequence QQYASVPVT (SEQ ID NO: 11),

the VL CDR2 comprises the amino acid sequence GASRLKT (SEQ ID NO: 10),and

the VL CDR1 comprises the amino acid sequence QASQSISSYLA (SEQ ID NO:9).

In certain embodiments, the antibody or antigen binding fragment mayadditionally comprise a light chain variable domain (VL) wherein:

the VL CDR3 consists of the amino acid sequence QQYASVPVT (SEQ ID NO:11),

the VL CDR2 consists of the amino acid sequence GASRLKT (SEQ ID NO: 10),and

the VL CDR1 consists of the amino acid sequence QASQSISSYLA (SEQ ID NO:9).

In certain embodiments, the antibody or antigen binding fragment thereofcomprises

-   -   a heavy chain variable domain (VH), wherein:        the VH CDR3 comprises the amino acid sequence YEWETVVVGDLMYEYEY        (SEQ ID NO: 13),        the VH CDR2 comprises the amino acid sequence RIDPEDAGTKYAQKFQG        (SEQ ID NO: 12), and        the VH CDR1 comprises the amino acid sequence SYYID (SEQ ID NO:        4); and    -   a light chain variable domain (VL), wherein:        the VL CDR3 comprises the amino acid sequence QQYASVPVT (SEQ ID        NO: 11),        the VL CDR2 comprises the amino acid sequence GASRLKT (SEQ ID        NO: 10), and        the VL CDR1 comprises the amino acid sequence QASQSISSYLA (SEQ        ID NO: 9).

In certain embodiments, the antibody or antigen binding fragment thereofcomprises

-   -   a heavy chain variable domain (VH), wherein:        the VH CDR3 consists of the amino acid sequence        YEWETVVVGDLMYEYEY (SEQ ID NO: 13),        the VH CDR2 consists of the amino acid sequence        RIDPEDAGTKYAQKFQG (SEQ ID NO: 12), and        the VH CDR1 consists of the amino acid sequence SYYID (SEQ ID        NO: 4); and    -   a light chain variable domain (VL), wherein:        the VL CDR3 consists of the amino acid sequence QQYASVPVT (SEQ        ID NO: 11),        the VL CDR2 consists of the amino acid sequence GASRLKT (SEQ ID        NO: 10), and        the VL CDR1 consists of the amino acid sequence QASQSISSYLA (SEQ        ID NO: 9).

In certain embodiments, the antibodies or antigen binding fragmentsinclude at least one heavy chain variable domain (VH) and/or at leastone light chain variable domain (VL) that is a humanised, germlined oraffinity variant of a camelid-derived VH or VL domain.

In certain embodiments, provided herein are antibodies or antigenbinding fragments thereof, which bind to the complex of human GARP andhuman TGF-β1, wherein the antibodies or antigen binding fragmentscomprise a heavy chain variable domain selected from the following:

-   -   (i) a VH comprising or consisting of the amino acid sequence of        SEQ ID NO: 14; or    -   (ii) a VH comprising or consisting of an amino acid sequence        having at least 90%, at least 95%, at least 97%, at least 98%,        or at least 99% identity to SEQ ID NO:14.

Alternatively or in addition, the antibodies or antigen bindingfragments may comprise a light chain variable domain (VL) selected fromthe following:

-   -   (i) a VL comprising or consisting of the amino acid sequence of        SEQ ID NO: 15; or    -   (ii) a VL comprising or consisting of an amino acid sequence        having at least 90%, at least 95%, at least 97%, at least 98%,        or at least 99% identity to SEQ ID NO: 15.

For embodiments wherein the domains of the antibodies or antigen bindingfragments are defined by a particular percentage sequence identity to areference sequence, the VH and/or VL domains may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only within the framework regions.

In a particular embodiment, provided herein are antibodies or antigenbinding fragments thereof, wherein the heavy chain variable domain (VH)comprises or consists of the amino acid sequence of SEQ ID NO: 14 andthe light chain variable domain (VL) comprises or consists of the aminoacid sequence of SEQ ID NO: 15.

In certain embodiments, the antibodies of the invention include the CH1domain, hinge region, CH2 domain and CH3 domain of a human antibody, inparticular human IgG1, IgG2, IgG3 or IgG4. In certain embodiments, theantibody includes the CH3 region of a human IgG4 and includes thesubstitution S228P in the CH3 domain.

The antibodies which bind the GARP-TGF-β1 complex may comprise at leastone full-length immunoglobulin heavy chain and/or at least onefull-length lambda or kappa light chain. In certain embodiments, theantibodies comprise a heavy chain comprising the amino acid sequence ofSEQ ID NO: 16 and a light chain comprising the amino acid sequence ofSEQ ID NO: 17. In certain embodiments, provided herein are monoclonalantibodies comprising a heavy chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 16. In certain embodiments, providedherein are monoclonal antibodies comprising a light chain with at least90%, at least 95%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 17. In certainembodiments, provided herein are monoclonal antibodies comprising aheavy chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 16, and a light chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 17.

For embodiments wherein the heavy and/or light chains of the antibodiesare defined by a particular percentage sequence identity to a referencesequence, the heavy chain and/or light chain may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only outside the CDR regions.

Unless otherwise stated in the present application, % sequence identitybetween two amino acid sequences may be determined by comparing thesetwo sequences aligned in an optimum manner and in which the amino acidsequence to be compared can comprise additions or deletions with respectto the reference sequence for an optimum alignment between these twosequences. The percentage of identity is calculated by determining thenumber of identical positions for which the amino acid residue isidentical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences. For example, itis possible to use the BLAST program, “BLAST 2 sequences” (Tatusova etal, “Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol Lett. 174:247-250), the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

The GARP-TGF-β1 antibodies or antigen binding fragments thereof providedherein may each exhibit one or more of the followingproperties/features:

-   -   the antibody or antigen binding fragment may cross-react with        the GARP-TGF-β complex of Cynomolgus origin;    -   the antibody or antigen binding fragment may bind to human        GARP-TGF-β1 with high affinity;    -   the antibody or antigen binding fragment may include a VH domain        and VL domain that when tested as a Fab fragment exhibit an        off-rate (K_(off)) for the complex of human GARP and TGF-β1 of        less than 5×10⁻⁴ s⁻¹;    -   the antibody or antigen binding fragment may include a VH domain        and VL domain that when tested as a Fab fragment exhibit an        off-rate (K_(off)) for the complex of human GARP and TGF-β1 in        the range 1×10⁻⁶ s⁻¹ to 5×10⁻⁴ s⁻¹;    -   the antibody or antigen binding fragment may include a VH domain        and VL domain that when tested as a mAb exhibit a K_(D) of less        than 1.7×10⁻⁹ M;    -   the antibody or antigen binding fragment may block release of        active TGF-β1 from regulatory T cells.

In further aspects, the invention also provides polynucleotide moleculeswhich encode the above-listed antibodies and antigen binding fragments,in addition to expression vectors comprising the polynucleotides, hostcells containing the vectors, and methods of recombinantexpression/production of the antibodies described herein.

In a still further aspect, the invention provides a pharmaceuticalcomposition comprising any one of the GARP-TGF-β1 antibodies or antigenbinding fragments thereof described herein, and a pharmaceuticallyacceptable carrier or excipient.

A still further aspect of the invention concerns methods of medicaltreatment using the above-listed GARP-TGF-β1 antibodies or antigenbinding fragments thereof, particularly in the prophylaxis and/ortreatment of TGF-β-related disorders. In certain embodiments, theinvention relates to methods of treatment using the GARP-TGF-β1antibodies or antigen binding fragments thereof, wherein the disease orcondition to be treated is selected from the group consisting ofinflammatory diseases, chronic infection, cancer, fibrosis,cardiovascular disease, cerebrovascular disease and neurodegenerativedisease. In certain embodiments, the GARP-TGF-β1 antibodies or antigenbinding fragments thereof are administered in combination with anothertreatment as part of a combination therapy. For example, the GARP-TGF-β1antibodies or antigen binding fragments thereof may be administered incombination with an immunotherapeutic agent, optionally animmunostimulatory antibody or a tumour vaccine.

These and other embodiments of the invention will be better appreciatedand understood when considered in conjunction with the followingdescription and the accompanying drawings. It should be understood,however, that the following description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the binding of latent TGF-β to GARP on thesurface of regulatory T cells. TGF-β is produced as a precursor,“pro-TGF-β” and undergoes cleavage to produce “latent-TGF-β”, a form inwhich the mature TGF-β dimer remains non-covalently associated with thelatency associated peptide (LAP) region of each polypeptide. It is thislatent form that binds to GARP on the surface of Treg cells. Integrinsαvβ6 and αvβ8 are thought to be responsible for mediating the release ofmature or “active TGF-β” from the cell surface. This active form can actin a paracrine fashion to bring about effects in a variety of targetcells, or can act as an autocrine mediator by binding to the TGF-βreceptor on Treg cells.

FIG. 2 shows target binding activity as measured by surface plasmonresonance (SPR) for antibodies 39B6 IgG1^(N297Q) and 39B6 IgG4^(S228P)over a 56-day period for samples stored at −20° C., 5° C. and 37° C. inPBS or PBSTween (PBSTw). The reference sample (−20° C.) was set as 100%binding activity at each time point.

FIGS. 3A and 3B show the results of testing the 39B6-A antibody variantsin an assay designed to monitor SMAD2 phosphorylation downstream ofTGF-β receptor activation. SMAD2 phosphorylation serves as a marker ofactivation of the TGF-β signalling pathway, following TGF-β binding toits receptor. If SMAD2 phosphorylation is reduced, TGF-β activity isinhibited. FIG. 3A: Western blots showing decreases in SMAD2phosphorylation in the presence of different concentrations of theGARP-TGF-β antibodies 39B6-A, 39B6-AVE, 39B6-AEE, 39B6-AYE, 39B6-ANR and39B6-ANK. FIG. 3B: Graphical representation of the data in (A) showingthe percentage inhibition of SMAD2 phosphorylation at different antibodyconcentrations.

FIG. 4 shows the results of testing the 39B6-A antibody variants in anassay designed to measure TGF-β activity via a luciferase reporter geneconjugated to a SMAD promoter. Graphs show percentage inhibition ofluminescence signal in the presence of different concentrations of theGARP-TGF-β antibodies LHG-10, 39B6-A, 39B6-AVE, 39B6-AEE, 39B6-AYE,39B6-ANR and 39B6-ANK.

FIG. 5 shows the percentage aggregate formation over a 56-day periodwith antibodies 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR stored at 5°C. and 37° C. Aggregate formation was monitored by size exclusionchromatography (SE-HPLC).

FIG. 6 shows the percentage fragment formation over a 56-day period withantibodies 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR stored at 37° C.Fragment formation was monitored by size exclusion chromatography(SE-HPLC).

FIG. 7 shows the percentage monomer area over a 56-day period withantibodies 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR stored at 5° C. and37° C. Monomer area was monitored by size exclusion chromatography(SE-HPLC).

FIGS. 8A-8D show the results of SOS-PAGE analysis of antibody samplesstored for 56 days at a reference temperature (−20° C.), at 5° C. and at37° C. FIG. 8A: 39B6-AVE. FIG. 8B: 39B6-AYE. FIG. 8C: 39B6-ANK. FIG. 8D:39B6-ANR. Markers appear at the centre of each gel. To the left of themarkers, the 3 samples are (i) Ref; (ii) 5° C.; and (iii) 37° C. samplestested under non-reducing conditions and to the right of the markers,the 3 samples are (i) Ref; (ii) 5° C.; and (iii) 37° C. samples testedunder reducing conditions.

FIG. 9 shows target binding activity as measured by SPR for antibodies39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR over a 56-day period forsamples stored at −20° C., 5° C. and 37° C. The reference sample (−20°C.) was set as 100% binding activity at each time point.

FIG. 10 shows the protein concentration (mg/ml) for samples ofantibodies 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR over a 56-dayperiod for samples stored at −20° C. (ref), 5° C. and 37° C.

FIG. 11 shows the results of SDS-PAGE analysis of antibody samples after10 freeze-thaw cycles. Markers appear at the centre of the gel. To theleft of the markers, the 4 samples are the samples analysed undernon-reducing conditions: (i) Ref for 39B6-AVE; (ii) Freeze-thaw samplefor 39B6-AVE; (iii) Ref for 39B6-AYE; and (iv) Freeze-thaw sample for39B6-AYE. To the right of the markers, the 4 samples are the samplesanalysed under reducing conditions: (i) Ref for 39B6-AVE; (ii)Freeze-thaw sample for 39B6-AVE; (iii) Ref for 39B6-AYE; and (iv)Freeze-thaw sample for 39B6-AYE.

FIG. 12 shows target binding activity as measured by SPR following 10freeze-thaw cycles for antibodies 39B6-AVE and 39B6-AYE. The referencesample (−20° C.) was set as 100% binding activity at each time point.

FIG. 13 shows the protein concentration (mg/ml) for samples ofantibodies 39B6-AVE and 39B6-AYE following 10 freeze-thaw cycles.

FIG. 14 shows target binding activity as measured by SPR followingthermal stability testing at temperatures ranging from 54.6° C. through71.4° C. for antibodies 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR. Thereference sample was set as 100% binding activity.

FIG. 15 shows the results of SDS-PAGE analysis of antibody samples after96 hours of rotation. Markers appear at the centre of the gel. To theleft of the markers, the 4 samples are the samples analysed undernon-reducing conditions: (i) Ref for 39B6-AVE; (ii) Rotated sample for39B6-AVE; (iii) Ref for 39B6-AYE; and (iv) Rotated sample for 39B6-AYE.To the right of the markers, the 4 samples are the samples analysedunder reducing conditions: (i) Ref for 39B6-AVE; (ii) Rotated sample for39B6-AVE; (iii) Ref for 39B6-AYE; and (iv) Rotated sample for 39B6-AYE.

FIG. 16 shows target binding activity as measured by SPR followingrotational stability testing for mAbs 39B6-AVE and 39B6-AYE. Thereference sample was set as 100% binding activity.

FIG. 17 shows the protein concentration (mg/ml) for samples of mAbs39B6-AVE and 39B6-AYE following rotational stability testing.

FIG. 18 shows the relative amount of deamidation and isomerization ofposition N95 in the antibodies 39B6-ANE, 39B6-ANR and 39B6-ANK over a56-day period. Antibodies 39B6-AVE and 39B6-AYE are not included becausethese antibodies have had residue “N95” removed from CDR3. Also shown isthe relative binding activity for mAbs 39B6-ANE, 39B6-AVE, 39B6-AYE,39B6-ANK and 39B6-ANR over the 56 d time course, for samples stored at37° C.

FIG. 19 shows the requirement for mature TGF-β in the binding of39B6-AYE (ARGX-115) to the GARP-TGF-β complex. ELISA plates were coatedwith either GARP or the anti-GARP Ab ARGX-115. For ELISA plates coatedwith GARP, a complex with either full-length latent TGF-β (includingboth the LAP and mature TGF-β regions) or a complex with recombinant LAPwas allowed to form by the addition of the relevant recombinant protein.For ELISA plates coated with ARGX-115, GARP was added and then eitherfull-length latent TGF-β or LAP was added. ARGX-115 was only able tobind GARP in the presence of full-length TGF-β. Binding of ARGX-115 tothe GARP-LAP complex did not occur. In contrast, an anti-LAP antibodywas able to bind to the GARP-LAP complex. This demonstrates therequirement for mature TGF-β for binding of ARGX-115 to the GARP-TGF-βcomplex.

FIG. 20 shows the ability of antibodies to neutralize TGF-β activationby the GARP-TGF-β complex with various mutant forms of TGF-β.Neutralizing activity of ARGX-115 was abrogated by mutation of R58 inLAP and K338 in mature TGF-β.

FIG. 21 shows a gradient PCR device program for the thermal stabilitystudy.

DETAILED DESCRIPTION A. Definitions

“GARP”—GARP (Glycoprotein A Repetitions Predominant) is a member of theleucine-rich repeat family of proteins. It is also called Leucine RichRepeat Containing 32 (LRRC32). GARP is an 80 kDa transmembrane proteinwith an extracellular region composed primarily of 20 leucine-richrepeats. The complete amino acid sequence of the human GARP proteintranscript variant 2 (GenBank Accession No. NP_001122394) is:

(SEQ ID NO: 33) MRPQILLLLALLTLGLAAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLAACCC VRRQKFNQQYKA.

“TGF-β”—TGF-β is a cytokine belonging to a superfamily of growthfactors. There are three distinct isoforms of TGF-β (TGF-β1, TGF-β2 andTGF-β3) encoded by three distinct genes, but the overall structures ofthe TGF-β isoforms are highly similar, with homologies in the order of70-80%. The term TGF-β, as used herein, is typically used to encompassall three different isoforms of the TGF-β cytokine, unless the contextindicates otherwise.

All three TGF-β isoforms are encoded as large protein precursors; TGF-β1(GenBank Accession No: NM_000660) contains 390 amino acids, and TGF-β2(GenBank Accession Nos: NM_001135599 and NM_003238) and TGF-β3 (GenBankAccession No: XM_005268028) each contain 412 amino acids. They each havean N-terminal signal peptide of 20-30 amino acids that is required forsecretion from a cell, a pro-region (named latency associated peptide orLAP), and a 112-114 amino acid C-terminal region that becomes the matureTGF-β molecule following its release from the pro-region by proteolyticcleavage. After proteolytic cleavage, LAP and mature TGF-β remainnon-covalently associated and form the “latent TGF-β” molecule. In thislatent form, mature TGF-β is prevented from binding to the TGF-βreceptor by LAP. To exert a signal, mature TGF-β must be released fromLAP. Mature TGF-β that is not associated to LAP is called active TGF-β,as it can bind to the TGF-β receptor and transduce a signal.

Full length TGF-β1 has the following amino acid sequence:

(SEQ ID NO: 34) MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNOHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

LAP has the following amino acid sequence:

(SEQ ID NO: 35) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR.

Mature TGF-β1 has the following amino acid sequence:

(SEQ ID NO: 36) ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS.

“GARP-TGF-β complex”—As used herein, the GARP-TGF-β complex means thenative complex that forms when latent TGF-β binds to GARP, particularlyGARP located on the surface of Treg cells. Although not specifiedthroughout, the “GARP-TGF-β complex,” or simply “GARP-TGF-β,” as usedherein, is intended to mean the complex between GARP and latent TGF-β.The binding of GARP to TGF-β, more specifically latent TGF-β, has beencharacterised at the molecular level, for example as reported in Wang etal. Mol Biol Cell. 2012 March; 23(6):1129-39. GARP forms a disulphidelinkage to the Cys4 of latent TGF-β and also associates with latentTGF-β through non-covalent interactions. There are 15 Cys residues inthe extracellular domain of GARP, and GARP uses Cys-192 and Cys-331 toform disulphide linkages to the two Cys4 residues of latent TGF-β. Itfollows that one GARP protein associates with one latent TGF-β dimer.

“Antibody” or “Immunoglobulin”—As used herein, the term “immunoglobulin”includes a polypeptide having a combination of two heavy and two lightchains whether or not it possesses any relevant specificimmunoreactivity. “Antibodies” refer to such assemblies which havesignificant specific immunoreactive activity to an antigen of interest(e.g. the complex of GARP and TGF-β). The term “GARP-TGF-β antibodies”is used herein to refer to antibodies which exhibit immunologicalspecificity for the complex of GARP and TGF-β1, particularly the humanGARP-TGF-β1 complex and in some cases species homologues thereof.Antibodies and immunoglobulins comprise light and heavy chains, with orwithout an interchain covalent linkage between them. Basicimmunoglobulin structures in vertebrate systems are relatively wellunderstood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody (IgG, IgM, IgA, IgD or IgE) that can be distinguishedbiochemically. All five classes of antibodies are within the scope ofthe present invention. The following discussion will generally bedirected to the IgG class of immunoglobulin molecules. With regard toIgG, immunoglobulins typically comprise two identical light polypeptidechains of molecular weight approximately 23,000 Daltons, and twoidentical heavy chains of molecular weight 53,000-70,000. The fourchains are joined by disulfide bonds in a “Y” configuration wherein thelight chains bracket the heavy chains starting at the mouth of the “Y”and continuing through the variable region.

The light chains of an antibody are classified as either kappa (κ) orlambda (λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells or genetically engineered host cells. In the heavychain, the amino acid sequences run from an N-terminus at the forkedends of the Y configuration to the C-terminus at the bottom of eachchain. Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA, IgD orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernible to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention.

As indicated above, the variable region of an antibody allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform the variable region that defines a three dimensional antigenbinding site. This quaternary antibody structure forms the antigenbinding site present at the end of each arm of the Y. More specifically,the antigen binding site is defined by three complementary determiningregions (CDRs) on each of the VH and VL chains.

“Binding Site”—As used herein, the term “binding site” comprises aregion of a polypeptide which is responsible for selectively binding toa target antigen of interest. Binding domains comprise at least onebinding site. Exemplary binding domains include an antibody variabledomain. The antibody molecules of the invention may comprise a singlebinding site or multiple (e.g., two, three or four) binding sites.

“Variable region” or “variable domain”—The terms “variable region” and“variable domain” are used herein interchangeably and are intended tohave equivalent meaning. The term “variable” refers to the fact thatcertain portions of the variable domains VH and VL differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its target antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called “hypervariableloops” in each of the VL domain and the VH domain which form part of theantigen binding site. The first, second and third hypervariable loops ofthe VLambda light chain domain are referred to herein as L1(λ), L2(λ)and L3(λ) and may be defined as comprising residues 24-33 (L1(λ),consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consistingof 3 residues) and 90-96 (L3(λ), consisting of 5 residues) in the VLdomain (Morea et al., Methods 20:267-279 (2000)). The first, second andthird hypervariable loops of the VKappa light chain domain are referredto herein as L1(κ), L2(κ) and L3(κ) and may be defined as comprisingresidues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues),49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).The first, second and third hypervariable loops of the VH domain arereferred to herein as H1, H2 and H3 and may be defined as comprisingresidues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (1-12,consisting of 3 or 4 residues) and 91-105 (H3, highly variable inlength) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both Vkappa and Vlambdaisotypes. The terms H1, H2 and H3 respectively refer to the first,second and third hypervariable loops of the VH domain, and encompasshypervariable loops obtained from any of the known heavy chain isotypes,including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”, as defined below.The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or31-35b (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of theantigen-binding site of antibodies. Structural analysis of antibodiesrevealed the relationship between the sequence and the shape of thebinding site formed by the complementarity determining regions (Chothiaet al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol.Biol, 215:175-182 (1990)). Despite their high sequence variability, fiveof the six loops adopt just a small repertoire of main-chainconformations, called “canonical structures”, These conformations arefirst of all determined by the length of the loops and secondly by thepresence of key residues at certain positions in the loops and in theframework regions that determine the conformation through their packing,hydrogen bonding or the ability to assume unusual main-chainconformations.

“CDR”—As used herein, the term “CDR” or “complementarity determiningregion” means the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat based on sequence comparisons.

TABLE 1 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H)CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3 95-10296-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclatureof Kabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

“Framework region”—The term “framework region” or “FR region” as usedherein, includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat definitionof CDRs). Therefore, a variable region framework is between about100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variabledomain and for the CDRs as defined by Kabat et al., framework region 1corresponds to the domain of the variable region encompassing aminoacids 1-30; framework region 2 corresponds to the domain of the variableregion encompassing amino acids 36-49; framework region 3 corresponds tothe domain of the variable region encompassing amino acids 66-94, andframework region 4 corresponds to the domain of the variable region fromamino acids 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light chainvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Constant region”—As used herein, the term “constant region” refers tothe portion of the antibody molecule outside of the variable domains orvariable regions. Immunoglobulin light chains have a single domain“constant region”, typically referred to as the “CL or CL1 domain”. Thisdomain lies C terminal to the VL domain. Immunoglobulin heavy chainsdiffer in their constant region depending on the class of immunoglobulin(γ, μ, α, δ, ε). Heavy chains γ, α and δ have a constant regionconsisting of three immunoglobulin domains (referred to as CH1, CH2 andCH3) with a flexible hinge region separating the CH1 and CH2 domains.Heavy chains μ and ε have a constant region consisting of four domains(CH1-CH4). The constant domains of the heavy chain are positioned Cterminal to the VH domain.

The numbering of the amino acids in the heavy and light immunoglobulinchains run from the N-terminus at the forked ends of the Y configurationto the C-terminus at the bottom of each chain. Different numberingschemes are used to define the constant domains of the immunoglobulinheavy and light chains. In accordance with the EU numbering scheme, theheavy chain constant domains of an IgG molecule are identified asfollows: CH1—amino acid residues 118-215; CH2—amino acid residues231-340; CH3—amino acid residues 341-446. In accordance with the Kabatnumbering scheme, the heavy chain constant domains of an IgG moleculeare identified as follows: CH1—amino acid residues 114-223; CH2—aminoacid residues 244-360; CH3—amino acid residues 361-477. The “hingeregion” includes the portion of a heavy chain molecule that joins theCH1 domain to the CH2 domain. This hinge region comprises approximately25 residues and is flexible, thus allowing the two N-terminal antigenbinding regions to move independently. Hinge regions can be subdividedinto three distinct domains: upper, middle, and lower hinge domains(Roux K. H. et al. J. Immunol. 161:4083-90 1998). Antibodies of theinvention comprising a “fully human” hinge region may contain one of thehinge region sequences shown in Table 2 below.

TABLE 2 Human hinge sequences IgG Upper hinge Middle hinge Lower hingeIgG1 EPKSCDKTHT CPPCP APELLGGP (SEQ ID NO: 37) (SEQ ID NO: 38)(SEQ ID NO: 39) IgG3 ELKTPLGDTTHT CPRCP(EPKSCDTPPPCPRCP)₃ APELLGGP(SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42) IgG4 ESKYGPP CPSCPAPEFLGGP (SEQ ID NO: 43) (SEQ ID NO: 44) (SEQ ID NO: 45) IgG2 ERKCCVECPPPCP APPVAGP (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48)

“Fragment”—The term “fragment”, as used in the context of antibodies ofthe invention, refers to a part or portion of an antibody or antibodychain comprising fewer amino acid residues than an intact or completeantibody or antibody chain. The term “antigen-binding fragment” refersto a polypeptide fragment of an immunoglobulin or antibody that bindsantigen or competes with intact antibody (i.e., with the intact antibodyfrom which they were derived) for antigen binding (i.e., specificbinding to the GARP-TGF-β complex). As used herein, the term “fragment”of an antibody molecule includes antigen-binding fragments ofantibodies, for example, an antibody light chain variable domain (VL),an antibody heavy chain variable domain (VH), a single chain antibody(scFv), a F(ab′)2 fragment, a Fab fragment, an Fd fragment, an Fvfragment, a one-armed (monovalent) antibody, diabodies, triabodies,tetrabodies or any antigen-binding molecule formed by combination,assembly or conjugation of such antigen binding fragments. The term“antigen binding fragment” as used herein is further intended toencompass antibody fragments selected from the group consisting ofunibodies, domain antibodies and nanobodies. Fragments can be obtained,e.g., via chemical or enzymatic treatment of an intact or completeantibody or antibody chain or by recombinant means.

“Conservative amino acid substitution”—A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an immunoglobulinpolypeptide may be replaced with another amino acid residue from thesame side chain family. In another embodiment, a string of amino acidscan be replaced with a structurally similar string that differs in orderand/or composition of side chain family members.

“Chimeric”—A “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. A chimeric protein may becreated, for example, by chemical synthesis, or by creating andtranslating a polynucleotide in which the peptide regions are encoded inthe desired relationship. Exemplary chimeric antibodies of the inventioninclude fusion proteins comprising camelid-derived VH and VL domains, orhumanised variants thereof, fused to the constant domains of a humanantibody, e.g. human IgG1, IgG2, IgG3 or IgG4.

“Valency”—As used herein the term “valency” refers to the number ofpotential target binding sites in a polypeptide. Each target bindingsite specifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen).

“Specificity”—The term “specificity” refers to the ability to bind(e.g., immunoreact with) a given target, e.g., a complex of GARP-TGF-β1.A polypeptide may be monospecific and contain one or more binding siteswhich specifically bind a target or a polypeptide may be multispecificand contain two or more binding sites which specifically bind the sameor different targets.

“Synthetic”—As used herein the term “synthetic” with respect topolypeptides includes polypeptides which comprise an amino acid sequencethat is not naturally occurring. For example, non-naturally occurringpolypeptides which are modified forms of naturally occurringpolypeptides (e.g., comprising a mutation such as an addition,substitution or deletion) or which comprise a first amino acid sequence(which may or may not be naturally occurring) that is linked in a linearsequence of amino acids to a second amino acid sequence (which may ormay not be naturally occurring) to which it is not naturally linked innature.

“Engineered”—As used herein the term “engineered” includes manipulationof nucleic acid or polypeptide molecules by synthetic means (e.g. byrecombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention are engineered, includingfor example, humanized and/or chimeric antibodies, and antibodies whichhave been engineered to improve one or more properties, such as antigenbinding, stability/half-life or effector function.

“Humanising substitutions”—As used herein, the term “humanisingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domain ofan antibody (for example a camelid-derived GARP-TGF-β1 antibody) isreplaced with an amino acid residue which occurs at an equivalentposition in a reference human VH or VL domain. The reference human VH orVL domain may be a VH or VL domain encoded by the human germline.Humanising substitutions may be made in the framework regions and/or theCDRs of the antibodies, defined herein.

“Humanised variants”—As used herein the term “humanised variant” refersto a variant antibody which contains one or more “humanisingsubstitutions” compared to a reference antibody, wherein a portion ofthe reference antibody (e.g. the VH domain and/or the VL domain or partsthereof containing at least one CDR) has an amino acid derived from anon-human species, and the “humanising substitutions” occur within theamino acid sequence derived from a non-human species.

“Germlined variants”—The term “germlined variant” is used herein torefer specifically to “humanised variants” in which the “humanisingsubstitutions” result in replacement of one or more amino acid residuespresent at a particular position (s) in the VH or VL domain of anantibody (for example a camelid-derived GARP-TGF-β1 antibody) with anamino acid residue which occurs at an equivalent position in a referencehuman VH or VL domain encoded by the human germline. It is typical thatfor any given “germlined variant”, the replacement amino acid residuessubstituted into the germlined variant are taken exclusively, orpredominantly, from a single human germline-encoded VH or VL domain. Theterms “humanised variant” and “germlined variant” are often usedinterchangeably herein. Introduction of one or more “humanisingsubstitutions” into a camelid-derived (e.g. llama derived) VH or VLdomain results in production of a “humanised variant” of the camelid(llama)-derived VH or VL domain. If the amino acid residues substitutedin are derived predominantly or exclusively from a single humangermline-encoded VH or VL domain sequence, then the result may be a“human germlined variant” of the camelid (llama)-derived VH or VLdomain.

“Affinity variants”—As used herein, the term “affinity variant” refersto a variant antibody which exhibits one or more changes in amino acidsequence compared to a reference antibody, wherein the affinity variantexhibits an altered affinity for the target antigen in comparison to thereference antibody. For example, affinity variants will exhibit achanged affinity for GARP-TGF-β, as compared to the reference GARP-TGF-βantibody. Preferably the affinity variant will exhibit improved affinityfor the target antigen, as compared to the reference antibody. Affinityvariants typically exhibit one or more changes in amino acid sequence inthe CDRs, as compared to the reference antibody. Such substitutions mayresult in replacement of the original amino acid present at a givenposition in the CDRs with a different amino acid residue, which may be anaturally occurring amino acid residue or a non-naturally occurringamino acid residue. The amino acid substitutions may be conservative ornon-conservative.

B. GARP-TGF-β1 Antibodies

The present invention relates to antibodies and antigen bindingfragments thereof, which specifically bind to the complex of GARP andTGF-β1, particularly the complex of human GARP and human TGF-β1. Theantibodies and antigen binding fragments of the present invention may bedefined with respect to structural and functional characteristics asdescribed herein.

Importantly, the GARP-TGF-β1 antibodies of the invention are improved ascompared with the GARP-TGF-β1 antibodies described previously, for thereason that they display improved stability. In particular, thestability of the GARP-TGF-β1 antibodies described herein is improved ascompared with antibodies having the heavy chain and light chain CDRsequences of the GARP-TGF-β1 reference antibodies LHG-10 and LHG-10.6,described in WO2015/015003 and WO2016/125017. This improvement instability is achieved without a significant decrease in the bindingaffinity of the antibodies for the GARP-TGF-β1 complex, as compared withthe reference LHG10 and LHG10.6 antibodies.

The antibodies of the present invention differ from the LHG-10 andLHG-10.6 GARP-TGF-β1 reference antibodies described previouslyparticularly with respect to the sequences of the heavy chain CDR2 andCDR3 sequences. More specifically, the LHG-10 and LHG-10.6 GARP-TGF-β1antibodies possess the heavy chain CDR2 sequence: RIDPEDGGTKYAQKFQG (SEQID NO: 5) and the heavy chain CDR3 sequence: NEWETVVVGDLMYEYEY (SEQ IDNO: 6), whereas the antibodies of the present invention comprise theheavy chain CDR2 sequence: RIDPEDAGTKYAQKFQG (SEQ ID NO: 12) and theheavy chain CDR3 sequence: YEWETVVVGDLMYEYEY (SEQ ID NO:13). Asdescribed and exemplified herein, the G55A and N95Y amino acidsubstitutions in the heavy chain CDR2 and CDR3 sequences, respectively,were found to improve antibody stability by reducing deamidation,isomerization and oxidation, whilst achieving a binding affinity for theGARP-TGF-β1 complex approximately equivalent to that of the referenceantibodies.

In a first aspect, the present invention provides antibodies or antigenbinding fragments thereof, which bind to a complex of GARP and TGF-β1,and comprise a heavy chain variable domain (VH) wherein:

the VH CDR3 comprises or consists of the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13),

the VH CDR2 comprises or consists of the amino acid sequenceRIDPEDAGTKYAQKFQG (SEQ ID NO: 12), and

the VH CDR1 comprises or consists of the amino acid sequence SYYID (SEQID NO: 4).

In certain embodiments, the antibodies or antigen binding fragmentsthereof additionally comprise a light chain variable domain (VL),wherein:

the VL CDR3 comprises or consists of the amino acid sequence QQYASVPVT(SEQ ID NO: 11),

the VL CDR2 comprises or consists of the amino acid sequence GASRLKT(SEQ ID NO: 10), and the VL CDR1 comprises or consists of the amino acidsequence QASQSISSYLA (SEQ ID NO: 9).

In certain embodiments, provided herein are antibodies or antigenbinding fragments thereof, which specifically bind the GARP-TGF-β1complex, wherein the antibodies or antigen binding fragments thereofcomprise at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein:

the VH CDR3 comprises or consists of the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13),

the VH CDR2 comprises or consists of the amino acid sequenceRIDPEDAGTKYAQKFQG (SEQ ID NO: 12),

the VH CDR1 comprises or consists of the amino acid sequence SYYID (SEQID NO: 4),

the VL CDR3 comprises or consists of the amino acid sequence QQYASVPVT(SEQ ID NO: 11),

the VL CDR2 comprises or consists of the amino acid sequence GASRLKT(SEQ ID NO: 10), and

the VL CDR1 comprises or consists of the amino acid sequence QASQSISSYLA(SEQ ID NO: 9).

In certain embodiments, provided herein are antibodies or antigenbinding fragments thereof, which specifically bind the GARP-TGF-β1complex, wherein the antibodies or antigen binding fragments thereofcomprise at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein:

the VH CDR3 consists of the amino acid sequence YEWETVVVGDLMYEYEY (SEQID NO: 13),

the VH CDR2 consists of the amino acid sequence RIDPEDAGTKYAQKFQG (SEQID NO: 12),

the VH CDR1 consists of the amino acid sequence SYYID (SEQ ID NO: 4),

the VL CDR3 consists of the amino acid sequence QQYASVPVT (SEQ ID NO:11),

the VL CDR2 consists of the amino acid sequence GASRLKT (SEQ ID NO: 10),and

the VL CDR1 consists of the amino acid sequence QASQSISSYLA (SEQ ID NO:9).

In certain embodiments, the antibodies and antigen binding fragments arerecombinant. In certain embodiments, the antibodies and antigen bindingfragments are monoclonal.

The term “antibody” herein is used in the broadest sense andencompasses, but is not limited to, monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, andmultispecific antibodies (e.g., bispecific antibodies), so long as theyexhibit the appropriate immunological specificity for the GARP-TGF-β1complex. The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes) on the antigen, each monoclonal antibody is directed againsta single determinant or epitope on the antigen.

The present invention also encompasses “antigen binding fragments” ofantibodies, and such fragments are defined elsewhere herein. Antibodyfragments typically comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, bi-specific Fab's, and Fvfragments, linear antibodies, single-chain antibody molecules, a singlechain variable fragment (scFv) and multispecific antibodies formed fromantibody fragments (see Holliger and Hudson, Nature Biotechnol.23:1126-36 (2005)).

The antibodies and antigen binding fragments of the present inventionmay exhibit high human homology. The level of homology with humansequence may be assessed across the length of the heavy chain variabledomain (VH) and/or across the length of the light chain variable domain(VL). In the context of the present invention, an antibody comprising aheavy chain variable domain (VH) and a light chain variable domain (VL)may be considered as having high human homology if the VH domains andthe VL domains, taken together, exhibit at least 90%, at least 92%, atleast 94%, or at least 96% amino acid sequence identity to the closestmatching human germline VH and VL sequences. In one embodiment the VHdomain of the antibody with high human homology may exhibit an aminoacid sequence identity or sequence homology of at least 90%, at least92%, at least 94%, or at least 96% with one or more human VH domainsacross the framework regions FR1, FR2, FR3 and FR4. In one embodimentthe VH domain of the antibody with high human homology may contain oneor more (e.g. 1 to 10) amino acid sequence mis-matches across theframework regions FR1, FR2, FR3 and FR4, in comparison to the closestmatched human VH sequence.

In another embodiment the VL domain of the antibody with high humanhomology may exhibit a sequence identity or sequence homology of atleast 90%, at least 92%, at least 94%, or at least 96% with one or morehuman VL domains across the framework regions FR1, FR2, FR3 and FR4. Inone embodiment the VL domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VL sequence.

Antibodies and antigen binding fragments of the present having highhuman homology may include antibodies comprising VH and VL domains ofnative non-human antibodies which exhibit sufficiently high % sequenceidentity to human germline sequences. In certain embodiments, theantibodies and antigen binding fragments of the invention are humanisedor germlined variants of non-human antibodies, for example antibodiescomprising VH and VL domains of camelid conventional antibodiesengineered so as to be humanised, or germlined variants of the originalantibodies.

The antibodies or antigen binding fragments thereof may comprise a heavychain variable domain (VH) comprising or consisting of the amino acidsequence of SEQ ID NO: 14 and optionally a light chain variable domain(VL) comprising or consisting of the amino acid sequence of SEQ ID NO:15.

In certain embodiments, the antibodies or antigen binding fragmentsthereof may comprise a heavy chain variable domain (VH) comprising theamino acid sequence of SEQ ID NO: 14. In certain embodiments, theantibodies or antigen binding fragments thereof may comprise a lightchain variable domain (VL) comprising the amino acid sequence of SEQ IDNO: 15.

In certain embodiments, the antibodies or antigen binding fragmentsthereof may comprise a heavy chain variable domain (VH) consisting ofthe amino acid sequence of SEQ ID NO: 14. In certain embodiments, theantibodies or antigen binding fragments thereof may comprise a lightchain variable domain (VL) consisting of the amino acid sequence of SEQID NO: 15.

In certain embodiments, the antibodies or antigen binding fragmentsthereof may comprise a heavy chain variable domain (VH) comprising theamino acid sequence of SEQ ID NO: 14 and a light chain variable domain(VL) comprising the amino acid sequence of SEQ ID NO: 15.

In certain embodiments, the antibodies or antigen binding fragmentsthereof may comprise a heavy chain variable domain (VH) consisting ofthe amino acid sequence of SEQ ID NO: 14 and a light chain variabledomain (VL) consisting of the amino acid sequence of SEQ ID NO: 15.

In certain embodiments, provided herein are monoclonal antibodies orantigen binding fragments thereof, comprising a heavy chain variabledomain and a light chain variable domain, the heavy chain variabledomain comprising a VH sequence with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 14 and/or the light chain variabledomain comprising a VL with at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceshown as SEQ ID NO: 15.

For embodiments wherein the domains of the antibodies or antigen bindingfragments are defined by a particular percentage sequence identity to areference sequence, the VH and/or VL domains may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only within the framework regions. In certainembodiments, the antibodies or antigen binding fragments comprisingheavy chain variable domains and/or light chain variable domains definedas having a particular percentage identity to SEQ ID NOs: 14 and 15,respectively, will have the following CDR sequences:

a VH CDR3 comprising or consisting of the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13),

a VH CDR2 comprising or consisting of the amino acid sequenceRIDPEDAGTKYAQKFQG (SEQ ID NO: 12),

a VH CDR1 comprising or consisting of the amino acid sequence SYYID (SEQID NO: 4),

a VL CDR3 comprising or consisting of the amino acid sequence QQYASVPVT(SEQ ID NO: 11),

a VL CDR2 comprising or consisting of the amino acid sequence GASRLKT(SEQ ID NO: 10), and

a VL CDR1 comprising or consisting of the amino acid sequenceQASQSISSYLA (SEQ ID NO: 9).

In non-limiting embodiments, the antibodies of the present invention maycomprise CH1 domains and/or CL domains (from the heavy chain and lightchain, respectively), the amino acid sequence of which is fully orsubstantially human. Where the antibody or antigen binding fragment ofthe invention is an antibody intended for human therapeutic use, it istypical for the entire constant region of the antibody, or at least apart thereof, to have fully or substantially human amino acid sequence.Therefore, one or more or any combination of the CH1 domain, hingeregion, CH2 domain, CH3 domain and CL domain (and CH4 domain if present)may be fully or substantially human with respect to its amino acidsequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanised or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. The invention alsocontemplates polypeptides comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required.

The presence of a “fully human” hinge region in the GARP-TGF-β1antibodies of the invention may be beneficial both to minimiseimmunogenicity and to optimise stability of the antibody.

As discussed elsewhere herein, it is contemplated that one or more aminoacid substitutions, insertions or deletions may be made within theconstant region of the heavy and/or the light chain, particularly withinthe Fc region. Amino acid substitutions may result in replacement of thesubstituted amino acid with a different naturally occurring amino acid,or with a non-natural or modified amino acid. Other structuralmodifications are also permitted, such as for example changes inglycosylation pattern (e.g. by addition or deletion of N- or O-linkedglycosylation sites).

The GARP-TGF-β1 antibodies may be modified within the Fc region toincrease binding affinity for the neonatal receptor FcRn. The increasedbinding affinity may be measurable at acidic pH (for example from aboutapproximately pH 5.5 to approximately pH 6.0). The increased bindingaffinity may also be measurable at neutral pH (for example fromapproximately pH 6.9 to approximately pH 7.4). By “increased bindingaffinity” is meant increased binding affinity to FcRn relative to theunmodified Fc region. Typically the unmodified Fc region will possessthe wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. Insuch embodiments, the increased FcRn binding affinity of the antibodymolecule having the modified Fc region will be measured relative to thebinding affinity of wild-type IgG1, IgG2, IgG3 or IgG4 for FcRn.

In certain embodiments, one or more amino acid residues within the Fcregion may be substituted with a different amino acid so as to increasebinding to FcRn. Several Fc substitutions have been reported thatincrease FcRn binding and thereby improve antibody pharmacokinetics.Such substitutions are reported in, for example, Zalevsky et al. (2010)Nat. Biotechnol. 28(2):157-9; Hinton et al. (2006) J Immunol.176:346-356; Yeung et al. (2009) J Immunol. 182:7663-7671; Presta L G,(2008) Curr. Op. Immunol. 20:460-470; and Vaccaro et al. (2005) Nat.Biotechnol. 23(10):1283-88, the contents of which are incorporatedherein in their entirety.

In certain embodiments, the GARP-TGF-β1 antibodies comprise a modifiedhuman IgG Fc domain comprising or consisting of the amino acidsubstitutions H433K and N434F, wherein the Fc domain numbering is inaccordance with EU numbering. In a further embodiment, the GARP-TGF-β1antibodies described herein comprise a modified human IgG Fc domaincomprising or consisting of the amino acid substitutions M252Y, S254T,T256E, H433K and N434F, wherein the Fc domain numbering is in accordancewith EU numbering.

In certain embodiments, the GARP-TGF-β1 antibodies comprise a modifiedhuman IgG Fc domain consisting of up to 2, up to 3, up to 4, up to 5, upto 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 20substitutions relative to the corresponding wild-type IgG sequence.

Depending on the intended use of the antibody, it may be desirable tomodify the antibody of the invention with respect to its bindingproperties to Fc receptors, for example to modulate effector function.For example cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp. Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922(1992). The invention also contemplates immunoconjugates comprising anantibody as described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). Fc regions may also beengineered for half-life extension, as described by Chan and Carter,Nature Reviews: Immunology, Vol. 10, pp 301-316, 2010, incorporatedherein by reference.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids.

In particular embodiments, the Fc region may be engineered such thatthere is no effector function. A GARP-TGF-β1 antibody having no Fceffector function may be particularly useful as a receptor blockingagent. In certain embodiments, the antibodies of the invention may havean Fc region derived from naturally-occurring IgG isotypes havingreduced effector function, for example IgG4. Fc regions derived fromIgG4 may be further modified to increase therapeutic utility, forexample by the introduction of modifications that minimise the exchangeof arms between IgG4 molecules in vivo. Fc regions derived from IgG4 maybe modified to include the S228P substitution.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the target antigen.Such carbohydrate modifications can be accomplished by, for example,altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen.

Also envisaged are variant GARP-TGF-β1 antibodies having an altered typeof glycosylation, such as a hypofucosylated antibody having reducedamounts of fucosyl residues or a fully or partially de-fucosylatedantibody (as described by Natsume et al., Drug Design Development andTherapy, Vol. 3, pp 7-16, 2009) or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC activity of antibodies, producingtypically 10-fold enhancement of ADCC relative to an equivalent antibodycomprising a “native” human Fc domain. Such carbohydrate modificationscan be accomplished by, for example, expressing the antibody in a hostcell with altered glycosylation enzymatic machinery (as described byYamane-Ohnuki and Satoh, mAbs 1:3, 230-236, 2009). Examples ofnon-fucosylated antibodies with enhanced ADCC function are thoseproduced using the Potelligent® technology of BioWa Inc.

Antibodies intended for human therapeutic use will typically be of theIgG, IgM, IgA, IgD, or IgE type, often of the IgG type, in which casethey can belong to any of the four sub-classes IgG1, IgG2a and b, IgG3or IgG4. Within each of these sub-classes it is permitted to make one ormore amino acid substitutions, insertions or deletions within the Fcportion, or to make other structural modifications, for example toenhance or reduce Fc-dependent functionalities.

In certain embodiments, the antibodies which specifically bindGARP-TGF-β1 comprise at least one full-length immunoglobulin heavy chainand/or at least one full-length lambda or kappa light chain, wherein theheavy chain comprises or consists of the amino acid sequence of SEQ IDNO:16 and the light chain comprises or consists of the amino acidsequence of SEQ ID NO:17.

In certain embodiments, the antibodies which specifically bindGARP-TGF-β1 comprise at least one full-length immunoglobulin heavy chainand/or at least one full-length lambda or kappa light chain, wherein theheavy chain comprises the amino acid sequence of SEQ ID NO:16 and thelight chain comprises the amino acid sequence of SEQ ID NO:17.

In certain embodiments, the antibodies which specifically bindGARP-TGF-β1 comprise at least one full-length immunoglobulin heavy chainand/or at least one full-length lambda or kappa light chain, wherein theheavy chain consists of the amino acid sequence of SEQ ID NO:16 and thelight chain consists of the amino acid sequence of SEQ ID NO:17.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO:16, and/or a light chain with at least 90%,at least 95%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence shown as SEQ ID NO:17.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO:16. In certain embodiments, provided hereinare monoclonal antibodies comprising a light chain with at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% sequence identityto the amino acid sequence shown as SEQ ID NO:17. In certainembodiments, provided herein are monoclonal antibodies comprising aheavy chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO:16, and a light chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO:17.

For embodiments wherein the chains of the antibodies are defined by aparticular percentage sequence identity to a reference sequence, theheavy chain and/or light chain may retain identical CDR sequences tothose present in the reference sequence such that the variation ispresent only outside the CDR regions. In particular, the antibodies orantigen binding fragments comprising heavy chains and/or light chainsdefined as having a particular percentage identity to SEQ ID NOs: 16 and17, respectively, may have the following CDR sequences:

a VH CDR3 comprising or consisting of the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13),

a VH CDR2 comprising or consisting of the amino acid sequenceRIDPEDAGTKYAQKFQG (SEQ ID NO: 12),

a VH CDR1 comprising or consisting of the amino acid sequence SYYID (SEQID NO: 4),

a VL CDR3 comprising or consisting of the amino acid sequence QQYASVPVT(SEQ ID NO: 11), and

a VL CDR2 sequence comprising or consisting of SEQ ID NO: 10 [GASRLKT]and

a VL CDR1 comprising or consisting of the amino acid sequenceQASQSISSYLA (SEQ ID NO: 9).

Binding to GARP-TGF-β1

The antibodies and antigen binding fragments of the present inventionbind to a complex of GARP and TGF-β1, particularly a complex of humanGARP and human TGF-β1. As explained elsewhere herein, GARP is atransmembrane protein expressed on the surface of regulatory T cells andacts as the receptor for the latent form of TGF-β. FIG. 1 includes aschematic representation of the complex that forms between GARP andlatent TGF-β at the cell surface of regulatory T cells.

The GARP-TGF-β1 complex to which the antibodies and antigen bindingfragments of the present invention bind is the native GARP-TGF-β1complex that forms at the cell surface between GARP and TGF-β1.

The antibodies and antigen binding fragments of the present inventionare characterised in that they bind to the complex of GARP-TGF-β1 but donot bind to GARP in the absence of TGF-β1 or latent TGF-β. Theantibodies and antigen binding fragments bind to GARP only in thepresence of TGF-β1. In particular, the antibodies and antigen bindingfragments bind to GARP only in the presence of latent TGF-β1.

Since the target antigen for the antibodies and antigen bindingfragments thereof of the present invention is a complex comprising twoseparate proteins, the epitope to which the antibodies and antigenbinding fragments bind is a conformational, as opposed to a linear,epitope. The conformational epitope comprises at least one residue fromGARP and at least one residue from latent TGF-β1. In preferredembodiments, the conformational epitope comprises at least one residuefrom GARP, at least one residue from the latency associated peptide(LAP) of latent TGF-β1 and at least one residue from mature TGF-β1.

The antibodies and antigen binding fragments of the present inventionmay bind to an epitope of a complex formed by human GARP and humanTGF-β1 wherein the epitope comprises at least one residue from GARPselected from Y137, S138, G139, T162 and R163 (with reference to SEQ IDNO: 33), and at least one residue from TGF-β1. In preferred embodiments,the epitope comprises at least residues Y137, S138, G139, T162 and R163of GARP (with reference to SEQ ID NO:33).

The epitope may comprise at least one residue from the TGF-β1polypeptide (SEQ ID NO: 34), optionally wherein the at least one residueis K338. The epitope may comprise at least one residue from the latencyassociated peptide (LAP) of TGF-β1 and at least one residue from matureTGF-β1. The epitope may comprise R58 of LAP and K338 of mature TGF-β1(with reference to SEQ ID NO: 34).

The antibodies and antigen binding fragments of the present inventionbind to a complex of GARP and TGF-β1. The antibodies and antigen bindingfragments of the present invention may additionally bind to a complex ofhuman GARP and human TGF-β2 and/or a complex of human GARP and humanTGF-β3.

In certain embodiments, antibodies and antigen binding fragments of theinvention bind to the complex of GARP and TGF-β1 with high affinity. Asused herein, the term “affinity” or “binding affinity” should beunderstood based on the usual meaning in the art in the context ofantibody binding, and reflects the strength and/or stability of bindingbetween an antigen and a binding site on an antibody or antigen bindingfragment thereof.

The binding affinity of an antibody or antigen binding fragment thereoffor its respective antigen can be determined experimentally usingtechniques known in the art. For example, SPR instruments such asBiacore™ measure affinity based on the immobilization of a targetprotein or antigen on a biosensor chip while the antibody or antibodyfragment is passed over the immobilized target under specific flowconditions. These experiments yield k_(on) and k_(off) measurements,which can be translated into K_(D) values, wherein K_(D) is theequilibrium constant for the dissociation of an antigen with an antibodyor fragment thereof. The smaller the K_(D) value, the stronger thebinding interaction between an antibody and its target antigen.

As noted above, the affinity of an antibody may be determined by SPR,for example using the protocol described elsewhere herein. The affinityof the antibody or antigen binding fragment for the GARP-TGF-β1 complex,as measured by SPR, may be determined using recombinantly expressedGARP-TGF-β1 complex, as described for example, in Example 2.

The GARP-TGF-β1 antibodies or antigen binding fragments thereof of theinvention may exhibit an off-rate (k_(off)) for the GARP-TGF-β1 complexof less than 7×10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 3×10⁻⁴ s⁻¹,less than 1.5×10⁻⁴ s⁻¹ when tested as a Fab. The GARP-TGF-β antibodiesor antigen binding fragments thereof of the invention may exhibit anoff-rate (k_(off)) for the complex of GARP-TGF-β1 in the range from1×10⁻⁶ s⁻¹ to 5×10⁻⁴ s⁻¹, preferably in the range from 1×10⁻⁶ s⁻¹ to3×10⁻⁴ s⁻¹, more preferably in the range from 1×10⁻⁵ s⁻¹ to 1.5×10⁻⁴s⁻¹.

The GARP-TGF-β1 antibodies of the invention may exhibit a K_(D) valueless than 5×10⁻⁹ M, less than 2×10⁻⁹ M. In preferred embodiments, theGARP-TGF-β1 antibodies of the invention exhibit a K_(D) value less than1.7×10⁻⁹ M.

In certain embodiments, the antibodies or antigen binding fragmentsdescribed herein that bind to the complex of GARP and TGF-β1 maycross-react with one or more species homologs of GARP and TGF-β, forexample GARP and TGF-β homologs of non-human primate origin.

In certain embodiments, the antibodies or antigen binding fragments ofthe present invention do not cross-react with the complex of GARP andTGF-β of murine origin. Alternatively or in addition, the antibodies orantigen binding fragments may bind to the GARP-TGF-β complex ofnon-human primate origin, particularly the GARP-TGF-β complex ofcynomolgus origin. The cross-reactivity with other species homologs canbe particularly advantageous in the development and testing oftherapeutic antibodies. For example, pre-clinical toxicology testing oftherapeutic antibodies is frequently carried out in primate speciesincluding but not limited to cynomolgus monkeys. Cross-reactivity withthese species homologs can therefore be particularly advantageous forthe development of antibodies as clinical candidates.

Improved Stability

The GARP-TGF-β1 antibodies of the invention are improved as comparedwith GARP-TGF-β1 antibodies described previously, for the reason thatthey display improved stability. In particular, the stability of theGARP-TGF-β1 antibodies is improved as compared with antibodies havingthe heavy chain and light chain CDR sequences of the GARP-TGF-β1reference antibody LHG10.6, described in WO2015/015003 andWO2016/125017.

The GARP-TGF-β antibody LHG10.6 possesses the following combination ofheavy chain variable domain and light chain variable domain CDRsequences:

heavy chain CDR3 consisting of (SEQ ID NO: 6) NEWETVVVGDLMYEYEY,heavy chain CDR2 consisting of (SEQ ID NO: 5) RIDPEDGGTKYAQKFQG,heavy chain CDR1 consisting of (SEQ ID NO: 4) SYYID,light chain CDR3 consisting of (SEQ ID NO: 11) QQYASVPVT,light chain CDR2 consisting of (SEQ ID NO: 10) GASRLKT, andlight chain CDR1 consisting of (SEQ ID NO: 9) QASQSISSYLA.

As reported elsewhere herein, it has been found that GARP-TGF-β1antibodies having the heavy chain and light chain CDR sequences ofLHG10.6 lack stability. In particular, germlined monoclonal antibodyvariants of LHG10.6 (referred to elsewhere herein as mAb 39B6 IgG4 and39B6 IgG1) were found to exhibit a trend towards lower target bindingactivity when stored at 37° C. in both PBS and PBS/Tween (see forexample, FIG. 2). This instability was attributed at least in part tothe isomerization and deamidation at position N95 of HCDR3, i.e. thefirst residue of heavy chain CDR3.

The GARP-TGF-β1 antibodies and antigen binding fragments of the presentinvention differ with respect to their CDR sequences, particularly theirheavy chain CDR sequences, such that their stability is improved. Inparticular, the heavy chain CDR2 (HCDR2 or VH CDR2) sequence is modifiedto include a G55A substitution, such that the HCDR2 sequence of theantibodies of the invention comprises the HCDR2 represented byRIDPEDAGTKYAQKFQG (SEQ ID NO: 12). In addition, the heavy chain CDR3(HCDR3 or VH CDR3) sequence is modified to include a N95Y substitution,such that the HCDR3 sequence of the antibodies of the inventioncomprises the HCDR3 represented by YEWETVVVGDLMYEYEY (SEQ ID NO: 13).The antibodies of the present invention do not undergo deamidation orisomerization. In addition, it has surprisingly been found that theGARP-TGF-β1 antibodies of the present invention are relatively resistantto oxidation. This resistance to deamidation, isomerization andoxidation correlates with improved stability of the antibodies of theinvention, particularly improved stability as measured at a temperatureof 37° C.

Furthermore, the antibodies and antigen binding fragments of theinvention are surprisingly advantageous because the modifications to theheavy chain CDR2 and CDR3 sequences, as compared with the referenceantibody LHG10.6, do not significantly decrease target binding activity.As exemplified herein, the GARP-TGF-β1 antibodies of the presentinvention are relatively stable at 37° C. and do not exhibit asignificant reduction in binding affinity for the GARP-TGF-β1 complex ascompared with the reference antibody LHG10.6 or a germlined variantthereof (39B6). As described elsewhere, in preferred embodiments, theGARP-TGF-β1 antibodies of the invention exhibit a K_(D) value less than1.7×10⁻⁹ M.

As reported herein, not all substitutions at position N95 of HCDR3 thatremove the Asn (N) residue are capable of improving antibody stabilitywhilst also retaining binding affinity for the GARP-TGF-β1 complex. Agermlined monoclonal antibody variant having an N95V substitution(referred to herein as 39B6-AVE) was found to be resistant todeamidation and isomerization, but underwent significant oxidation uponstorage for 28 days at 37° C. The binding activity of this N95V antibodyvariant was also found to decrease significantly over a 56-day period ofstorage at 37° C. The inventors also found that bulky substitutions atadjacent position 96 in the heavy chain (i.e. the second residue ofHCDR3) were incapable of improving stability and retaining high affinityantigen binding activity. As exemplified herein, the inventors testedtwo germlined monoclonal antibody variants including the substitutionsE96K and E96R in the HCDR3 domain. The E96K variant (referred to hereinas 39B6-ANK) did not undergo oxidation, but was subject to deamidationand isomerization over a 28-day period at 37° C. and underwent asignificant decrease in binding activity over a 56-day period. The E96Rvariant (referred to herein as 39B6-ANR) underwent significant oxidationand deamidation over a 28-day period at 37° C. and underwent a decreasein binding activity over a 56-day period.

In preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein

the VH CDR3 comprises or consists of the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13),

the VH CDR2 comprises or consists of the amino sequenceRIDPEDAGTKYAQKFQG (SEQ ID NO: 12),

the VH CDR1 comprises or consists of the amino acid sequence SYYID (SEQID NO: 4),

the VL CDR3 comprises or consists of the amino acid sequence QQYASVPVT(SEQ ID NO: 11),

the VL CDR2 comprises or consists of the amino sequence GASRLKT (SEQ IDNO: 10), and

the VL CDR1 comprises or consists of the amino acid sequence QASQSISSYLA(SEQ ID NO: 9).

In certain preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein

the VH CDR3 comprises the amino acid sequence YEWETVVVGDLMYEYEY (SEQ IDNO: 13),

the VH CDR2 comprises the amino sequence RIDPEDAGTKYAQKFQG (SEQ ID NO:12),

the VH CDR1 comprises the amino acid sequence SYYID (SEQ ID NO: 4),

the VL CDR3 comprises the amino acid sequence QQYASVPVT (SEQ ID NO: 11),

the VL CDR2 comprises the amino sequence GASRLKT (SEQ ID NO: 10), and

the VL CDR1 comprises the amino acid sequence QASQSISSYLA (SEQ ID NO:9).

In certain preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein the VH CDR3 consists of theamino acid sequence YEWETVVVGDLMYEYEY (SEQ ID NO: 13),

the VH CDR2 consists of the amino sequence RIDPEDAGTKYAQKFQG (SEQ ID NO:12),

the VH CDR1 consists of the amino acid sequence SYYID (SEQ ID NO: 4),

the VL CDR3 consists of the amino acid sequence QQYASVPVT (SEQ ID NO:11),

the VL CDR2 consists of the amino sequence GASRLKT (SEQ ID NO: 10), and

the VL CDR1 consists of the amino acid sequence QASQSISSYLA (SEQ ID NO:9).

These antibodies preferably exhibit a K_(D) value less than 1.7×10⁻⁹ M.

In particularly preferred embodiments of the invention, the antibodieswhich specifically bind the GARP-TGF-β1 complex and exhibit improvedstability comprise a heavy chain variable domain (VH) comprising orconsisting of the amino acid sequence of SEQ ID NO: 14 and optionally alight chain variable domain (VL) comprising or consisting of the aminoacid sequence of SEQ ID NO: 15.

In particularly preferred embodiments of the invention, the antibodieswhich specifically bind the GARP-TGF-β1 complex and exhibit improvedstability comprise a heavy chain variable domain (VH) comprising theamino acid sequence of SEQ ID NO: 14 and a light chain variable domain(VL) comprising the amino acid sequence of SEQ ID NO: 15.

In particularly preferred embodiments of the invention, the antibodieswhich specifically bind the GARP-TGF-β1 complex and exhibit improvedstability comprise a heavy chain variable domain (VH) consisting of theamino acid sequence of SEQ ID NO: 14 and a light chain variable domain(VL) consisting of the amino acid sequence of SEQ ID NO: 15.

In further preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise a heavy chain comprising or consisting of the amino acidsequence of SEQ ID NO: 16 and optionally a light chain comprising orconsisting of the amino acid sequence of SEQ ID NO: 17.

In further preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:17.

In further preferred embodiments of the invention, the antibodies whichspecifically bind the GARP-TGF-β1 complex and exhibit improved stabilitycomprise a heavy chain consisting of the amino acid sequence of SEQ IDNO: 16 and a light chain consisting of the amino acid sequence of SEQ IDNO: 17.

Polynucleotides Encoding GARP-TGF-β Antibodies

The invention also provides polynucleotide molecules comprising one ormore nucleotide sequences encoding the GARP-TGF-β1 antibodies or antigenbinding fragments of the invention, expression vectors containing saidnucleotide sequences of the invention operably linked to regulatorysequences which permit expression of the antibodies or fragments thereofin a host cell or cell-free expression system, and host cells orcell-free expression systems containing said expression vectors.

In certain embodiments, the heavy chain variable domain and/or the lightchain variable domain of the GARP-TGF-β1 antibodies or antigen-bindingfragments according to the present invention are encoded by first andsecond polynucleotide sequences, wherein the first and secondpolynucleotide sequences comprise the amino acid sequences of SEQ IDNOs: 18 and 19, respectively. In certain embodiments, thepolynucleotides encoding the GARP-TGF-β1 antibodies of the invention maycomprise variant sequences which encode functional VH or VL domains of aGARP-TGF-β1 antibody. The variant sequences encoding VH domains mayexhibit at least 80%, 85%, 90%, 95%, 97% or 99% sequence identity whenoptimally aligned to SEQ ID NO: 18, and the variant sequences encodingVL domains may exhibit at least 80%, 85%, 90%, 95%, 97% or 99% sequenceidentity when optimally aligned to SEQ ID NO: 19.

In certain embodiments, the heavy chain and/or the light chain of theGARP-TGF-β1 antibodies or antigen-binding fragments according to thepresent invention are encoded by first and second polynucleotidesequences, wherein the first and second polynucleotide sequencescomprise the amino acid sequences of SEQ ID NOs: 20 and 21,respectively. In certain embodiments, the polynucleotides encoding theGARP-TGF-β1 antibodies of the invention may comprise variant sequenceswhich encode heavy chains or light chains of a GARP-TGF-β1 antibody. Thevariant sequences encoding heavy chains may exhibit at least 80%, 85%,90%, 95%, 97% or 99% sequence identity when optimally aligned to SEQ IDNO: 20, and the variant sequences encoding light chains may exhibit atleast 80%, 85%, 90%, 95%, 97% or 99% sequence identity when optimallyaligned to SEQ ID NO: 21.

In this context, % sequence identity between two polynucleotidesequences may be determined by comparing these two sequences aligned inan optimum manner and in which the polynucleotide sequence to becompared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide residue is identicalbetween the two sequences, by dividing this number of identicalpositions by the total number of positions in the comparison window andby multiplying the result obtained by 100 in order to obtain thepercentage of identity between these two sequences. For example, it ispossible to use the BLAST program, “BLAST 2 sequences” (Tatusova et al,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol Lett. 174:247-250) available atncbi.nlm.nih.gov/gorf/bl2, the parameters used being those given bydefault (in particular for the parameters “open gap penalty”: 5, and“extension gap penalty”: 2; the matrix chosen being, for example, thematrix “BLOSUM 62” proposed by the program), the percentage of identitybetween the two sequences to be compared being calculated directly bythe program.

Polynucleotide molecules encoding the antibodies of the inventioninclude, for example, recombinant DNA molecules. The terms “nucleicacid”, “polynucleotide” or “polynucleotide molecule” as usedinterchangeably herein refer to any DNA or RNA molecule, either single-or double-stranded and, if single-stranded, the molecule of itscomplementary sequence. In discussing nucleic acid molecules, a sequenceor structure of a particular nucleic acid molecule may be describedherein according to the normal convention of providing the sequence inthe 5′ to 3′ direction. In some embodiments of the invention, nucleicacids or polynucleotides are “isolated.” This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or non-human hostorganism. When applied to RNA, the term “isolated polynucleotide” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been purified/separated from other nucleic acids with which it wouldbe associated in its natural state (i.e., in cells or tissues). Anisolated polynucleotide (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

For recombinant production of an antibody according to the invention, arecombinant polynucleotide encoding it may be prepared (using standardmolecular biology techniques) and inserted into a replicable vector forexpression in a chosen host cell or a cell-free expression system.Suitable host cells may be prokaryote, yeast, or higher eukaryote cells,specifically mammalian cells. Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen. Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581; ATCCCRL 8287) or NS0 (HPA culture collections no. 85110503); monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM'sPERC-6 cell line. Expression vectors suitable for use in each of thesehost cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line. Whole human beings into which an expression vectorencoding an antibody or antigen binding fragment according to theinvention has been introduced are explicitly excluded from thedefinition of a “host cell”.

Antibody Production

In a further aspect, the invention also provides a method of producingantibodies of the invention which comprises culturing a host cell (orcell-free expression system) containing one or more polynucleotides(e.g., an expression vector) encoding the antibody under conditionswhich permit expression of the antibody, and recovering the expressedantibody. This recombinant expression process can be used for largescale production of antibodies, including GARP-TGF-β1 antibodiesaccording to the invention, including monoclonal antibodies intended forhuman therapeutic use. Suitable vectors, cell lines and productionprocesses for large scale manufacture of recombinant antibodies suitablefor in vivo therapeutic use are generally available in the art and willbe well known to the skilled person.

Pharmaceutical Compositions

The scope of the invention includes pharmaceutical compositionscontaining one or a combination of GARP-TGF-β1 antibodies of theinvention, or antigen-binding fragments thereof, formulated with one ormore pharmaceutically acceptable carriers or excipients. Suchcompositions may include one or a combination of (e.g., two or moredifferent) GARP-TGF-β1 antibodies. Techniques for formulating monoclonalantibodies for human therapeutic use are well known in the art and arereviewed, for example, in Wang et al., Journal of PharmaceuticalSciences, Vol. 96, pp 1-26, 2007, the contents of which are incorporatedherein in their entirety.

In certain embodiments, the pharmaceutical compositions are formulatedfor administration to a subject via any suitable route of administrationincluding but not limited to intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, epidural, nasal, oral, rectal, topical,inhalational, buccal (e.g., sublingual), and transdermal administration.

Pharmaceutically acceptable excipients that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (for example sodiumcarboxymethylcellulose), polyethylene glycol, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Therapeutic Utility of GARP-TGF-β1 Antibodies

The antibodies and antigen binding fragments of the present inventionmay be used in methods of treatment, wherein a subject in need thereofis administered a therapeutically effective amount of a GARP-TGF-β1antibody or antigen binding fragment thereof. In certain embodiments,the antibodies and antigen binding fragments of the present inventionmay be used in methods of treatment, wherein a human subject in needthereof is administered a therapeutically effective amount of aGARP-TGF-β1 antibody or antigen binding fragment thereof. Providedherein are antibodies or antigen binding fragments thereof, which bindto the complex of human GARP and TGF-β1, for use as medicaments.

The antibodies and antigen binding fragments of the invention bind tothe GARP-TGF-β complex on regulatory T cells and can block active TGF-βproduction or release. Therefore, in a further aspect of the invention,provided herein are methods for treating subjects having or suspected ofhaving TGF-β-related disorders. Such methods involve administering to asubject in need thereof a therapeutically effective amount of aGARP-TGF-β1 antibody of the invention.

Exemplary TGF-β-related disorders include, but are not limited to,inflammatory diseases, chronic infection, cancer, fibrosis,cardiovascular diseases, cerebrovascular disease (e.g. ischemic stroke),and neurodegenerative diseases. In certain embodiments, a TGF-β-relateddisorder is chronic infection. In certain embodiments, a TGF-β-relateddisorder is cancer.

For use in administration to a subject, the antibodies and antigenbinding fragments of the invention may be formulated as pharmaceuticalcompositions. The compositions may be administered orally, parenterally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “administration” as used hereinincludes without limitation subcutaneous, intravenous, intraperitoneal,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intratumoral, and intracranialinjection or infusion techniques.

Sterile injectable forms of the compositions may be aqueous or anoleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

Schedules and dosages for administration of the antibody or antigenbinding fragment thereof in the pharmaceutical compositions can bedetermined in accordance with known methods for these types of products.For example, an antibody present in a pharmaceutical composition of thisinvention can be supplied at a concentration of 10 mg/mL in either 100mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulatedfor intravenous (IV) administration in 9.0 mg/mL sodium chloride, 7.35mg/mL sodium citrate dihydrate, 0.7 in g/mL polysorbate 80, and SterileWater for Injection. The pH is adjusted to 6.5.

For clinical use, the antibody or antigen binding fragment can beadministered to a subject in one or more doses. For parenteral routes ofadministration, a single dose of the antibody or antigen bindingfragment can be, for example, about 0.01 to about 100 mg/kg body weight.In an embodiment, a single dose of the antibody or antigen bindingfragment can be, for example, about 0.1 to about 50 mg/kg body weight.In an embodiment, a single dose of the antibody or antigen bindingfragment can be, for example, about 1 to about 20 mg/kg body weight. Forrepeated dosing, individual doses can be administered by the same ordifferent routes of administration. Also for repeated dosing, individualdoses can be the same or different. For example, a first or loading dosemay be more than a subsequent dose. Also for repeated dosing, individualdoses can be administered on a fixed schedule or on an adjustable orvariable schedule based, for example, on a subject's clinical conditionor clinical response. Also for repeated dosing, individual dosestypically can be administered once daily, once every other day, onceevery 3, 4, 5, 6, or 7 days, once weekly, once biweekly, once everythree or four weeks, or once every other month. Other schedules are alsocontemplated by the invention.

It will be appreciated that these doses and schedules are exemplary andthat an optimal schedule and regimen can be determined by taking intoaccount such factors as the particular antibody or antigen bindingfragment to be administered, the disease or disorder to be treated, thesize, age and condition of the subject to be treated, the route ofadministration, other therapies being administered to the subject, andthe affinity and tolerability of the particular antibody. Such factorsand dosing considerations can be determined in one or more clinicaltrials.

The present invention also provides methods for boosting the immunesystem in a subject in need thereof, comprising administering to asubject a therapeutically effective amount of an antibody or antigenbinding fragment of the invention. The present invention also providesmethods for inhibiting the immune suppressive function of human Tregs ina subject in need thereof, comprising administering to the subject atherapeutically effective amount of an antibody or antigen bindingfragment of the invention.

The present invention also provides methods for the treatment of cancerusing GARP-TGF-β1 antibodies and antigen binding fragments of theinvention. Such methods may involve reducing immunosuppression in thetumor environment in a subject in need thereof.

For embodiments wherein the GARP-TGF-β1 antibodies or antigen bindingfragments thereof are for use in methods of treating cancer, theantibodies or antigen binding fragments thereof may be administered incombination with one or more additional treatments for cancer, forexample one or more immunotherapeutic agent(s).

For embodiments wherein the GARP-TGF-β1 antibodies are administered incombination with an immunotherapeutic agent, said immunotherapeuticagent may be a tumor vaccine. Alternatively, the immunotherapeutic agentmay be an immunostimulatory antibody. Without wishing to be bound bytheory, the antibodies of the invention will likely improve the efficacyof the immunotherapeutic agent by preventing or alleviating anyimmunosuppression. In certain embodiments, the combination with animmunotherapeutic agent may exhibit a synergistic effect.

Various cancers can be treated in accordance with the methods describedherein including but not limited to adrenocortical carcinoma, analcancer, bladder cancer, brain tumor, glioma, breast carcinoma, carcinoidtumor, cervical cancer, colon carcinoma, endometrial cancer, esophagealcancer, extrahepatic bile duct cancer, Ewing's tumor, extracranial germcell tumor, eye cancer, gall bladder cancer, gastric cancer, germ celltumor, gestational trophoblastic tumor, head and neck cancer,hypopharyngeal cancer, islet cell carcinoma, kidney cancer, laryngealcancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer,lymphoma, melanoma, mesothelioma, Merkel cell carcinoma, metastaticsquamous head and neck cancer, myeloma, neoplasm, nasopharyngeal cancer,neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovariancancer, pancreatic cancer, sinus and nasal cancer, parathyroid cancer,penile cancer, pheochromocytoma, pituitary cancer, plasma cell neoplasm,prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma,salivary gland cancer, skin cancer, Kaposi's sarcoma, T-cell lymphoma,soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer,or Wilms' tumor.

Suitable tumor antigens for use as tumor vaccines known in the artinclude for example: (a) cancer-testis antigens such as NY-ESO-1, SSX2,SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, forexample, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6,and MAGE-12 (which can be used, for example, to address melanoma, lung,head and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b)mutated antigens, for example, p53 (associated with various solidtumors, e.g., colorectal, lung, head and neck cancer), p21/Ras(associated with, e.g., melanoma, pancreatic cancer and colorectalcancer), CD 4 (associated with, e.g., melanoma), MUM 1 (associated with,e.g., melanoma), caspase-8 (associated with, e.g., head and neckcancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701,beta catenin (associated with, e.g., melanoma), TCR (associated with,e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g.,chronic myelogenous leukemia), triosephosphate isomerase, IA 0205,CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example, Galectin4 (associated with, e.g., colorectal cancer), Galectin 9 (associatedwith, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g.,chronic myelogenous leukemia), WT 1 (associated with, e.g., variousleukemias), carbonic anhydrase (associated with, e.g., renal cancer),aldolase A (associated with, e.g., lung cancer), PRAME (associated with,e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lungand ovarian cancer), alpha-fetoprotein (associated with, e.g.,hepatoma), SA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), andcarcinoembryonic antigen (associated with, e.g., breast cancer, lungcancer, and cancers of the gastrointestinal tract such as colorectalcancer), (d) shared antigens, for example, melanoma-melanocytedifferentiation antigens such as MART-I/Melan A, gp100, MC1R,melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase relatedprotein-1/TRPI and tyrosinase related protein-2/TRP2 (associated with,e.g., melanoma), (e) prostate associated antigens such as PAP, PSA,PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer, (f)immunoglobulin idiotypes (associated with myeloma and B cell lymphomas,for example), and (g) other tumor antigens, such as polypeptide- andsaccharide-containing antigens including (i) glycoproteins such assialyl Tn and sialyl Le<x> (associated with, e.g., breast and colorectalcancer) as well as various mucins; glycoproteins may be coupled to acarrier protein (e.g., MUC-1 may be coupled to LH); (ii)lipopolypeptides (e.g., MUC-1 linked to a lipid moiety); (iii)polysaccharides (e.g., Globo H synthetic hexasaccharide), which may becoupled to a carrier proteins (e.g., to KLH), (iv) gangliosides such asGM2, GM12, GD2, GD3 (associated with, e.g., brain, lung cancer,melanoma), which also may be coupled to carrier proteins (e.g., KLH).Other tumor antigens include pi 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens, including E6 and E7, hepatitis B and Cvirus antigens, human T-cell lymphotrophic virus antigens, TSP-180,pl85erbB2, pl80erbB-3, c-met, mn-23H I, TAG-72-4, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, p 16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72,beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242,CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein/cyclophilin C-associated protein), TAAL6, TAG72, TLP,TPS, and the like. Suitable immunostimulatory antibodies include, butare not limited to: anti-CTLA-4, anti-PD1, anti-PDL1 and anti-KIRantibodies.

In certain embodiments, the methods for treating cancer describedherein, comprise administering to the subject the antibody or antigenbinding fragment of the invention prior to, concurrent to and/or afterthe administration of another anti-cancer agent or cancer treatment,such as chemotherapy treatment.

The present invention also includes methods for preventing infectiousdiseases by administering antibodies or antigen binding fragments of theinvention so as to improve the efficacy of vaccination strategies. Forexample, the methods of the invention may include the prevention ofinfectious diseases such as HIV, malaria, or Ebola by combinedadministration of antibodies or antigen binding fragments as describedherein together with vaccines particular to these diseases.

Kits

In a further aspect, the present invention provides a kit comprising atleast one GARP-TGF-β1 antibody or antigen binding fragment of theinvention.

The term “kit” is intended to mean any manufacture (e.g., a package or acontainer) comprising at least one reagent, e.g., an antibody or antigenbinding fragment of the invention, for specifically binding theGARP-TGF-β1 complex. The kit may be promoted, distributed, or sold as aunit for performing the methods of the present invention. Furthermore,any or all of the kit reagents may be provided within containers thatprotect them from the external environment, such as in sealedcontainers. The kits may also contain a package insert describing thekit and methods for its use.

EXAMPLES

The invention will be further understood with reference to the followingnon-limiting examples.

Example 1: Germlining Antibody LHG-10.6

The production and characterisation of antibody “LHG-10” is described inInternational patent applications WO2015/015003 and WO2016/125017, thecontents of which are incorporated herein in their entirety. AntibodyLHG-10 was raised by immunizing llamas with HEK293E cells overexpressingthe human GARP-TGF-β1 complex, and was identified by selecting andscreening for GARP-TGF-β1 Fabs. A light chain shuffling approach (asdescribed for example, in International patent applicationWO2014/033252) was used to improve the affinity of the monoclonalantibody LHG-10, and a variant “LHG-10.6” (also referred to herein as17H5) was identified as having improved binding characteristics. The VHand VL sequences of LHG-10 and the Vκ shuffled variant LHG-10.6 areshown in Table 3.

TABLE 3 SEQ ID NO: VH EVQLVQPGAELRNSGASVKVSCKASGYRFTSYYIDWVRQAPGQGLEWM 1domain of GRIDPEDGGTKYAQKFQGRVTFTADTSTSTAYVELSSLRSEDTAVYYCAR LHG-10NEWETVVVGDLMYEYEYWGQGTQVTVSS and LHG10.6 VLDIQMTQSPSSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPKLLIYGA 2 domain ofSRLQTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYDSLPVTFGQG LHG-10 TKVELK VLDIQMTQSPSSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPNILIYGA 3 domain ofSRLKTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYASVPVTFGQG LHG10.6 TKVELK

The 17H5 antibody was found to have 88.5% identity with the closesthuman germline sequence (X92343|IGHV1-46*01) across the VH domain and86.2% identity with the closest human germline sequence(X59315|IGKV1-39*01) across the VL domain. This antibody was subjectedto germlining according to a 3-step method:

-   -   1—Assembly of a gene library by using overlapping        oligonucleotides to synthetically generate the variable light        (VL) and variable heavy (VH) chain encoding genes via PCR.    -   2—Cloning of this gene library into a vector (pCB13) containing        the human constant heavy (CH1) and constant light kappa (C_(K))        chain encoding genes (library construction).    -   3—Selection of the functional Fabs using phage display and        affinity selection.        1. Library Construction

The VH of 17H5 was compared with the closest human germline sequence andframework residues deviating from the human germline were identified.These framework residues were allowed to mutate to the residue presentin the human V-region, whilst also maintaining in the library theoriginal residue. The 12 VH residues that were allowed to mutate totheir human counterparts are shown in the table below. In addition tothese germlining mutations, an isomerization site within CDR2 (D54;1055) and an oxidation site in CDR3 (M100f) were allowed to mutate.These sites are also shown in the table below.

TABLE 4 Targeted residues in the 17H5 VH domain Position Camelid aaMutated aa Probability Clones 1 E Q 0.5 2 7 P S 0.5 2 11 L V 0.5 2 12 RK 0.5 2 13 N K 0.5 2 14 S P 0.5 2 28 R T 0.5 2 54 (HCDR2) D E 0.5 2 55(HCDR2) G A 0.5 2 69 F M 0.5 2 71 A R 0.5 2 78 A V 0.5 2 80 V M 0.5 2100f (HCDR3) M L/T 0.33 3 108 Q L 0.5 2

For the Vk of 17H5, a comparison between the closest human germlinesequence and the framework sequences led to the identification of 11residues to be targeted. These sites are shown in the table below.

TABLE 5 Targeted residues in the 17H5 VL domain Position Camelid aaMutated aa Probability Clones 11 L V 0.5 2 42 Q K 0.5 2 45 N K 0.5 2 46I L 0.5 2 70 S D 0.5 2 77 G S 0.5 2 79 E Q 0.5 2 80 A P 0.5 2 83 A F 0.52 84 G A 0.5 2 106 L I 0.5 2

The theoretical sizes of the 17H5 germlined library are shown in Table6.

TABLE 6 17H5 size of the VH library 5 × 10⁴ size of the VL library 2 ×10³ size of the final library 1 × 10⁸2. Construction of the Gene Library

The germlined libraries were created by gene assembly. Synthetic genesfor VH and VL (two sets) based on this design were generated by PCRbased assembly (Stemmer et al., Gene (1995) 164: 49-530). Overlappingoligonucleotides with specific mutations on certain positions wereassembled by PCR. The nucleotides were degenerated to encode the humanand the llama amino acid. This was done to prevent complete loss ofbinding in case the llama residues were critical for stability, foldingor high affinity binding.

The synthetic genes of the 17H5 Vκ and VH libraries were first cloned inpCB13-CK1, a phagemid vector with the human Ck and CH1 domains,respectively. After construction of these VkCk and VHCH1 sub-libraries,the final libraries were made by ligation of the heavy chain insert intothe light chain library. A colony PCR was carried out to determine theinsert percentage and clones were sent for sequencing to ensure that alldesired mutations were present in the library. The characteristics ofthe different libraries are described in the table below.

TABLE 7 Characteristics of the 17H5 germlined libraries 17H5 size of theVH library 1E5 size of the VL library 1E3 size of the final library 1E8% insert 85%

The germlining libraries were found to be of good quality and could beused for selections.

3. Selection of Fabs with High Human Identity

Phage display was used to select for Fabs with high affinity for thehuman GARP-TGF-β1 complex, as described previously in WO2015/015003, thecontents of which are incorporated herein in their entirety. In short, amicrotiter plate was coated O/N at 4′C with the GARP-TGF-β1 complex andthe day after, Fab expressing phages were incubated for 2 hours in wellscoated with different concentrations of complex. Plates were washed andspecific phage were eluted with trypsin and used for infection of TG1cells. Details of the selection conditions are listed in Table 8.

TABLE 8 Details of the selection rounds for the germlined library of17H5 Phage huGARP-TGF-β1 Washing Washing Round input (μl) [ng/well] timeantigen 1 10 1000-100-10 — — 2 1 1000-100-10 — — 3 1 1000-100-10  2hours  20 × huGT 4 1 1000-100-10 24 hours  20 × huGT 5 1 1000-100-10 72hours  20 × huGT 6 1 1000-100-10  1 week 100 × huGT

After the first round of selection, enrichment was only observed over acoating of an irrelevant protein at 10 μg/ml. In round 2, the enrichmentwas 10-fold higher, while the amount of input phage was 10-fold lower.Starting from round 3, the stringency was increased by increasing theduration of the washing and by adding an excess of target in solution. Alarge excess of soluble target is solution was added to preventrebinding of the dissociated phage. Even after the 6^(th) round, with a1-week off-rate washing and a 100-fold excess of soluble target,enrichment was still observed.

4. Screening of Fabs with High Human Identity

Masterplates were picked from selection round 2, 3, 4, 5 and 6 (48clones for each round). Periplasmic extracts were prepared and off-rateswere determined by SPR. All clones were DNA-sequenced and amino acidsequences were deduced thereof. Clones with an identity to humangermline Ig V-region above 92% are summarized in Table 9. The off-ratesof these clones were measured as periplasmic extracts on SPR, andcompared to the off-rate of Fab 17H5.

TABLE 9 Binding affinity of clones with more than 92% total humanidentity Off-rate % Identity % Identity % Overall Isomerisation siteRisk oxidation Fab (10⁻⁴ s⁻¹) VH VK Identity D54; G55 (HCDR2) M100f(HCDR3) 17H5 2.01 88.5 86.2 87.4 wt (D54; G55) wt (M100f) *39A12 6.0695.4 96.2 95.8 E54; G55 T100f *39A5 1.9 94.2 96.2 95.2 wt wt 39H6 4.493.1 96.2 94.7 wt wt 40H3 2.47 94.2 95.0 94.6 wt wt 39A10 1.77 94.2 95.094.6 wt wt *39B6 1.05 95.4 93.7 94.6 wt wt 39E6 8.61 96.5 92.5 94.5 wtwt 40G4 2.07 93.1 95.0 94.1 wt wt 38C3 2.32 95.4 92.5 94.0 wt wt 39G36.62 95.4 92.5 94.0 E54; G55 T100f 39G9 9.47 95.4 92.5 94.0 E54; G55T100f 38F3 2.03 91.9 95.0 93.5 D54; A55 wt *38F2 4.08 94.2 92.5 93.4D54; A55 T100f *39B10 2.36 95.4 91.2 93.3 E54; G55 wt 39C6 7.89 95.491.2 93.3 E54; G55 T100f 39G6 7.79 94.2 91.2 92.7 wt L100f 38C5 1.6990.8 93.7 92.3 E54; G55 wt 38B2 2.86 93.1 91.2 92.2 wt T100f *5 Fabsthat were reformatted as full IgG1

The 5 clones showing an overall human identity of >93% and an off-ratecomparable to that of the parental 17H5 Fab were recloned as full humanIgG1 antibodies. The CDR, VH and VL sequences of these antibodies areshown in Tables 10, 11 and 12.

TABLE 10 VH CDR sequences for GARP-TGF-β1 Abs SEQ ID SEQ ID SEQ ID CDR1NO: CDR1 NO: CDR1 NO: 39B6 SYYID 4 RIDPEDGGTKYAQKFQG 5 NEWETVVVGDLMYEYEY6 39A12 SYYID 4 RIDPEEGGTKYAQKFQG 7 NEWETVVVGDLTYEYEY 32 39A5 SYYID 4RIDPEDGGTKYAQKFQG 5 NEWETVVVGDLMYEYEY 6 38F2 SYYID 4 RIDPEDAGTKYAQKFQG 8NEWETVVVGDLTYEYEY 32 39B10 SYYID 4 RIDPEEGGTKYAQKFQG 5 NEWETVVVGDLMYEYEY6

TABLE 11 VL CDR sequences for GARP-TGF-β1 Abs SEQ ID SEQ ID SEQ ID CDR1NO: CDR2 NO: CDR3 NO: 39B36 QASQSISSYLA 9 GASRLKT 10 QQYASVPVT 11 39A12QASQSISSYLA 9 GASRLKT 10 QQYASVPVT 11 39A5 QASQSISSYLA 9 GASRLKT 10QQYASVPVT 11 38F2 QASQSISSYLA 9 GASRLKT 10 QQYASVPVT 11 39B10QASQSISSYLA 9 GASRLKT 10 QQYASVPVT 11

TABLE 12 Variable domain sequences for GARP-TGF-β1 Abs SEQ ID SEQ IDClone VH NO: VL NO: 39B6 QVQLVQPGAEVRKPGASVKVSCKASGYRFTSYYI 22DIQMTQSPSSLSASVGDRVTITCQASQSISSYL 23 DWVRQAPGQGLEWMGRIDPEDGGTKYAQKFQGAWYQQKPGQAPKILIYGASRLKTGVPSRFSGS RVTMTADTSTSTVYVELSSLRSEDTAVYYCARNEGSGTSFTLTISSLEPEDAATYYCQQYASVPVTF WETVVVGDLMYEYEYWGQGTLVTVSS GQGTKVEIK39A12 EVQLVQPGAEVKKPGASVKVSCKASGYRFTSYYID 24DIQMTQSPSSLSASVGDRVTITCQASQSISSYL 25 WVRQAPGQGLEWMGRIDPEEGGTKYAQKFQGRVAWYQQKPGQAPKILIYGASRLKTGVPSRFSGS TFTADTSTSTVYVELSSLRSEDTAVYYCARNEWETGSGTDFTLTISSLQAEDFATYYCQQYASVPVT VVVGDLTYEYEYWGQGTLVTVSS FGQGTKVEIK 39A5QVQLVQPGAELRNPGASVKVSCKASGYRFTSYYI 26 DIQMTQSPSSLSASLGDRVTITCQASQSISSYL27 DWVRQAPGQGLEWMGRIDPEDGGTKYAQKFQG AWYQQKPGQAPKLLIYGASRLKTGVPSRFSGRVTFTRDTSTSTVYMELSSLRSEDTAVYYCARNE SGSGTDFTLTISSLQPEDAATYYCQQYASVPVWETVVVGDLMYEYEYWGQGTLVTVSS TFGQGTKVEIK 38F2QVQLVQPGAELKKPGASVKVSCKASGYRFTSYYID 28 DIQMTQSPSSLSASVGDRVTITCQASQSISSYL29 WVRQAPGQGLEWMGRIDPEDAGTKYAQKFQGRV AWYQQKPGKAPNLLIYGASRLKTEVPSRFSGSTFTADTSTSTVYVELSSLRSEDTAVYYCARNEWET GSGTDFTLISGLEPEDAGTYYCQQYASVPVTFVVVGDLTYEYEYWGQGTLVTVSS GQGTKVEIK 39B10EVQLVQSGAELKKPGASVKVSCKASGYRFTSYYID 30 DIQMTQSPSSLSASLGDRVTITCQASQSISSYL31 WVRQAPGQGLEWMGRIDPEEGGTKYAQKFQGRV AWYQQKPGQAPNILIYGASRLKTGVPSRFSGSTMTADTSTSTAYMELSSLRSEDTAVYYCARNEWE GSGTDFTLTISGLEAEDFATYYCQQYASVPVTTVVVGDLMYEYEYWGQGTLVTVSS FGQGTKVEIK5. In Vitro Characterisation of mAbs with High Human Framework ResidueIdentity

The five germlined clones reformatted to human IgG1 were produced bytransient transfection in HEK 293E cells. All the antibodies were testedfor binding to the human GARP-TGF-β1 complex by SPR and showed bindingaffinity similar to the non-germlined parental clone (KD 2-5 times lowerthan 17H5). Changing residue M100f in the CDR3 to a threonine decreasedthe K_(D)≈5-fold. Binding properties and characteristics of thegermlined 17H5 Abs are listed in Table 13.

TABLE 13 Binding properties and characteristics of the germlined 17H5Abs risk % % % fold isomerisation oxidation Identity Identity IdentitymAb K_(D) (M) difference site D54; G55 M100f VH VK overall 17H5 1.00E−101.0 wt (DG) wt (M) 88.5 86.2 87.3 39B6 1.67E−10 1.7 wt (DG) wt (M) 95.493.7 94.5 39B10 2.62E−10 2.6 EG wt (M) 95.4 91.2 93.3 39A5 2.98E−10 3.0wt (DG) wt (M) 94.2 96.2 95.2 38F2 4.80E−10 4.8 DA T 94.2 92.5 93.339A12_T 5.05E−10 5.1 EG T 95.4 96.2 95.86. Stability of Germlined mAb 39B6

Clone 39B6 was selected as the lead germlined clone based on its bindingcharacteristics. This antibody was produced in two effector functiondeficient formats, hIgG1^(N297Q) and hIgG4^(S228P), and stability testswere carried out. Prior to the stability testing, the affinity of theeffector deficient 39B6 antibodies for GARP-TGF-β1 was tested byBiacoreT200 using a CM5 chip coated with GARP-TGF-β1 complex at 750RU(Table 14) or coated with the antibodies at 1000RU (Table 15).

TABLE 14 mAb Format K_(D) (M) Fold-difference LHG10.6 IgG1-N297Q8.19E−10 1 39B6 IgG1-N297Q 8.63E−11 0.1 39B6 IgG1-N297Q 3.13E−10 0.4

TABLE 15 mAb Format K_(D) (M) Fold-difference LHG10.6 IgG1-N297Q5.34E−10 1 39B6 IgG1-N297Q 1.23E−09 2.3 39B6 IgG1-N297Q 1.02E−09 1.9

The thermo-tolerance of the 39B6 antibodies was tested using thefollowing set-up.

-   -   mAb sample preparation: 1 ml of the mAbs (39B6-IgG1 and        39B6-IgG4), fresh prepared stock solution (5 mg/ml), were put in        2 ml glass screw cap glass vials and stored at 5° C. and 37° C.        Vials were checked immediately for absence of particles.    -   PBS negative control: aliquots of 1 ml filtered Dulbecco's PBS        were prepared in 2 ml glass vials as the PBST negative control.        Vials were checked immediately for absence of particles.    -   PBSTw negative control: aliquots of 1 ml filtered Dulbecco's PBS        containing 0.02% Tween80 (Sigma) were prepared in 2 ml glass        vials as the PBSTw negative control. Vials were checked        immediately for absence of particles.    -   High-aggregation control: 1 ml aliquots of an in-house antibody        were prepared with abundant visible aggregation in 2 ml glass        vials.    -   Reference sample: for each antibody, 60 μl mAb aliquots were        prepared in 500 μl sterile, PCR tubes, labeled and stored at        −20° C.

The stability of the antibodies stored at different temperatures andunder different conditions (PBS versus PBSTw) was monitored over a56-day time course. The effect of storage on target binding activity asmeasured by SPR (Biacore™) is shown in FIG. 2. As can be seen from theresults presented, the 39B6 antibody (in both effector deficientformats) exhibited a trend towards lower target binding activity in bothPBS and PBS/Tween when stored at 37° C. In contrast, the samples storedat 5° C. did not display a significant loss in target binding activityover the time-course.

Example 2: Development of GARP-TGF-β1 Antibodies with Improved Stability

2.1 Production of 39B6 Variants

The decrease in target binding activity noted above for antibody 39B6was found to be linked to deamidation occurring at positions 95-96 of VHCDR3. As such, further work was carried out to improve the stability of39B6.

First, this clone was subjected to modification of the VH chain in theCDR2 region to introduce a G55A substitution (39B6-A). The VH and VLregions of 39B6-A were recloned into the human IgG4^(S228P) backbonei.e. the effector function deficient human antibody format.

In an attempt to improve the stability of antibody 39B6-A IgG4^(S228P),five variants were generated having mutations at positions 95 or 96 ofCDR3 of the VH domain. These variants are shown below. All variants wereproduced in the effector function deficient format IgG4^(S228P).

TABLE 16 39B6-A Variants Variant Position 95 Position 96 39B6-AVE V E*39B6-AYE Y E* 39B6-ANK N* K 39B6-ANR N* R 39B6-AEE E E* *residue alreadypresent in 39B6-A

The antibodies were filtered and concentrated to 5 mg/ml and stored inDulbecco's PBS with 0.02% Tween80 (Sigma). The Tween80 was needed tostabilize the antibody formulations.

The affinity of all antibodies (including 39B6-A) for GARP-TGF-β1 wastested by BiacoreT200 using a CM5 chip coated with GARP-TGF-β1 complexat 150RU (Table 17) or coated with the antibodies at 200RU (Table 17)

TABLE 17 mAb K_(D) (M) Fold-difference 39B6-A 9.91E−10 1.0 39B6-AVE8.94E−10 0.9 39B6-AYE 7.63E−10 0.8 39B6-AEE 5.79E−09 5.8 39B6-ANK8.80E−10 0.9 39B6-ANR 8.84E−10 0.9 LHG10.6 5.17E−10 0.5

TABLE 18 mAb K_(D) (M) Fold-difference 39B6-A 1.33E−09 1.0 39B6-AVE1.29E−09 1.0 39B6-AYE 1.62E−09 1.2 39B6-AEE 1.02E−07 76.7 39B6-ANK2.02E−09 1.5 39B6-ANR 1.94E−09 1.5

The potency to block the release of active TGF-β by human Tregs wasanalyzed with all variants by determining the level of SMAD2phosphorylation of CD3/CD28 stimulated human Tregs in the presence ofthe mAbs. As shown in FIG. 3, antibodies 39B6-AYE, 39B6-ANK and 39B6-ANRhad a similar potency in the SMAD2 phosphorylation assay compared to theoriginal 39B6-A. The variants 39B6-AEE and 39B6-AVE showed a clear lossin potency.

Similar results were obtained using the GAGA-luc assay where 293T-hGARPcells were transiently transfected with a reporter plasmid in whichFirefly luciferase is under the control of a SMAD responding promoter(GAGA-luc). Antibodies 39B6-AYE, 39B6-ANK and 39B6-ANR had a similarpotency in the luciferase assay compared to the original 39B6-A. Thevariants 39B6-AEE and 39B6-AVE showed a clear loss in potency (see FIG.4).

2.2 Stability Studies

With the exception of 39B6-AEE, the variants were tested for stabilityusing different approaches as described below.

For each of the different stability studies, the antibodies were testedusing one or more of the following techniques:

-   -   Visual inspection    -   Size Exclusion-HPLC    -   SDS-PAGE (reducing and non-reducing)    -   Target binding affinity on SPR    -   Protein concentration        The protocols for these techniques are described below.        Protocol for Visual Inspection

Samples were blinded and scored for the presence of visible particles byvisual inspection of the vials by three analysts. Samples were allowedto reach room temperature for 30 minutes before inspection. Thefollowing scoring system was used for the assessment:

A: sample is clear, no particles visible

B: very few particles

C: moderate presence of particles

D: abundant particles observed

f: fibers

Protocol for Size-Exclusion Chromatography (SE-HPLC)

System, Column and Sample Preparation

The column used throughout the study was an Xbridge Protein BEH SEC 200A(3.5 μm, 7.8*30 mm, Waters) which is routinely stored in 20% EtOH. AnXbridge Protein BEH 200A pre-guard column was coupled to the analyticalcolumn (3.5 μm, 7.8*3 mm; Waters). Before each utilization, the columnwas equilibrated with Dulbecco's PBS for 5CV at least, and before beingremoved from the system, it was cleaned with 5CV of LC-grade water. Allsolvents were filtered and degassed before utilization.

The chromatographic system used was an Agilent 1260 Infinity, equippedwith a quaternary pump, automatic injector, on-line degasser and a DADdetector. The column was not kept in a thermostated compartment andsamples were analyzed at room temperature. The detector was set towavelengths 280 and 214 nm simultaneously (reference wavelength at 360nm with a cut-off of 100 nm). Aggregation monitoring was followed onchannel 214 nm. Data acquisition was done with the Chemstation software(Agilent).

Sample analysis was done by transferring 25 μl of all original samplesdirectly from the 5 mg/ml concentration under aseptic conditions. Theywere spun down at 2000 rcf for 1 min and 20 μl was carefully transferredinto screw-cap, amber, glass vials. The injection volume for eachcondition was 5 μl (25 μg), at a flow rate of 0.7 ml PBS/min for 25 min.Each injection was accompanied with a needle wash with LC-grade waterand seal washing took place at the end of each sample.

Every time point sequence was initiated with 2×PBS injections (blank; 5μl injection volume), followed by 2 μl BEH 200 Standard Protein mixtureinjection (Waters) and a known-aggregation mAb sample. Every twelvesamples analyzed, a blank injection was done. Each analytical sequencewas terminated as initiated but in the reverse order (known-aggregationcontrol mAb, BEH standard protein mixture and 2× blank).

Method Used for Determination of % Aggregation and % Monomer Area

The following protocol was used:

-   -   Integrate all chromatograms with the method ARGX-115 (basic        technical parameters; tangent skim standard mode, slope        sensitivity 2.0, height reject 1.7, area reject 1.0, peak width:        0.02)    -   Verify that all peaks are integrated in a way that coincides        with the previous analyses obtained    -   Export the integration results in a PDF and Excel file    -   Calculate the % total aggregation as the summary of areas of all        peaks eluting before the monomer peak divided by the total area        of the injection and multiply by 100    -   Also calculate the % monomer area by dividing the monomer area        by the total area and multiply by 100. This % monomer area        reflects if there are insoluble aggregates present in the sample    -   Keep detailed records to monitor the performance, efficiency and        resolution of the analytical column (i.e. peak symmetry, monomer        area, total area, reproducibility of retention times etc.)        Protocol for SDS-PAGE        Sample Preparation

Sample analysis was done by transferring 25 μl of all original samplesat 5 mg/ml in 100 μl PBS/0.02% Tween80 (abbreviated as PBSTw throughoutthis study) under aseptic conditions in order to generate anintermediate concentration at 1 mg/ml, for SDS-PAGE (reducing andnon-reducing), binding affinity on SPR and protein concentrationassessment. 4-20% tris glycine, mini-protean, stain-free, pre-cast gelswere used for SDS-PAGE analysis of the samples (Biorad). A batch of4×-concentrated loading dye, with or without reducing agent DTT, wasprepared and aliquoted to −20° C. Routinely, 5 μl was taken from theintermediate dilution at 1 mg/ml and then, 5 μl of 4× concentratedloading dye (+/−DDT) was added together with 10 μl of mQ. The finalquantity for each condition was 5 μg. Samples were boiled for 10 min at95° C. and then loaded (20 μl) to the pre-cast gels. Electrophoresistook place for 35 min in a tris-glycine buffer system under a constantvoltage of 200 V. Blue staining (Gentaur) followed for 1 h. All gelswere de-stained with mQ water for at least 1 h.

Determination of % Full Length Ab

The intensity of the different bands was determined on the Odyssey v3.0Li-Cor system by scanning the protein gels to one-channel detection (700nm) under the settings: focus offset 0.5 mm and “high” quality. Thebrightness and contrast for each analysis were set to 50% with a linearmanual parameter of 5.

The method was as follows:

-   -   Scan the gel and verify that all bands on gel are surrounded by        tight rectangles    -   Choose “export” in the settings in order to obtain the raw        intensity of all bands in an Excel format    -   Calculate the % raw intensity of each band present in a lane as        the raw intensity of the band divided by the total raw intensity        of the lane and multiply by 100    -   For the non-reducing conditions, the % full length Ab is defined        as the % raw intensity of the band which corresponds to the ˜100        kD band    -   For the reducing conditions, the % full length Ab is defined as        the summary of the % raw intensity of the bands which correspond        to the ˜25-35 kD and ˜55 kD bands        Protocol for Target Binding Activity Measurement in Biacore 3000        Sample Preparation

From the intermediate concentration of all samples at 1 mg/ml describedabove, an extra 1/250 dilution was done for SPR analysis to yield afinal concentration of 4 μg/ml. This 1/250 dilution took place for allconditions in two steps: a) 5 μl (1 mg/ml)+120 μl HBS-EP (SPR buffer)and b) an extra 10× dilution (15 μl+135 μl HBS-EP). For each antibodyand each time point, a separate standard curve was prepared, startingfrom a −80° C. frozen sample. This was analyzed together with thestability samples to determine the slope of each curve after generalfitting of the standards. These slopes were used to calculate thepercent activity of the stability samples by setting the referencesample at 100%.

Determination of % Activity on Biacore

To determine the target binding activity in Biacore, a CM5 chip wascoated with ˜4000 RU human GARP-TGF-β complex. Flow was set to 30 μl/minwith a “kinject” injection mode and two regeneration injections (1 mMNaCl, 2.5 mM glycine pH 1.5) with a 5 min interval.

The method was carried out as follows:

-   -   Open the curves in the BIAEVAL program and select value ‘2-1’        (1: blank, 2 hGARP-TGF-β1)    -   Select one curve at the time for plot overlay    -   Delete the regeneration part of the curve    -   Select the baseline just before the injection and transform the        Y-axis for ‘zero at median of selection’    -   Select ‘General Fit’, starting from 120 sec after injecting and        ending at 155 sec    -   Plot the standards in Excel and determine the slope of the curve        for each antibody    -   These values are converted to a percent activity by setting the        reference (sample stored at −20° C.) at 100%        Protocol for Protein Concentration

For the determination of the protein content of each condition, theNanoDrop system was used by measuring the absorbance at 280 nm of eachsample (2 μl). The system was blanked with PBS and the proteindetermination was done by measuring in triplicates the absorbance at 280nm of each sample (intermediate dilution at 1 mg/ml). All valuesobtained at 280 nm were divided by the factor 1.51. A PBS blankmeasurement was done after each different condition. All data werereported as average value of the three measurements for each condition.

0.2.1 Temperature Stability Study

To monitor the stability of the antibodies under different temperaturestorage conditions, the following set-up was followed:

-   -   mAb sample preparation: 1 ml of the miAbs (39B6-AVE, 39B6-AYE,        39B6-ANK and 39B6-ANR), fresh prepared stock solution (5 mg/ml),        were put in 2 ml glass screw cap glass vials and stored at 5° C.        and 37° C. Vials were checked immediately for absence of        particles.    -   PBSTw negative control: aliquots of 1 ml filtered Dulbecco's PBS        containing 0.02% Tween80 (Sigma) were prepared in 2 ml glass        vials as the PBSTw negative control. Vials were checked        immediately for absence of particles.    -   High-aggregation control: 1 ml aliquots of an in-house antibody        were prepared with abundant visible aggregation in 2 ml glass        vials.    -   Reference sample: for each antibody, 60 μl mAb aliquots were        prepared in 500 μl sterile, PCR tubes, labeled and stored at        −20° C.

At different time points, samples of the mAbs were taken from thestorage conditions and tested for:

-   -   Visual inspection    -   SE-HPLC    -   SDS-PAGE (reducing and non-reducing)    -   target binding affinity on SPR    -   protein concentration        Visual Inspection

The results for visual inspection are shown in Table 19 below. All mAbswere in PBSTw, which should give a stabilizing effect.

TABLE 19 Results of visual inspection of mAb samples of 39B6-AVE,39B6-AYE, 39B6-ANK and 39B6-ANR over a 56-day period under storageconditions of 5° C. and 37° C. 0 d 1 d 7 d 14 d 28 d 56 d Sample Storingconditions: 5° C. 39B6-AVE A-B-B A-B-B Af-B-B Af-B-B Af-B-A B-Bf-Af39B6-AYE A-A-A A-A-A A-A-B Af-A-B Af-Af-B Af-Bf-A 39B6-ANK A-A-A A-A-BAf-B-B Af-A-B B-A-C B-A-B 39B6-ANR B-A-A B-B-B B-Bf-Bf C-Bf-Bf C-Bf-CfC-Bf-C High C-C-C C-C-C C-D-D D-D-D D-D-D D-D-D aggregation controlPBSTw Af-A-A Af-A-A A-B-B Af-A-B Af-A-A Af-A-A Storing conditions: 37°C. 39B6-AVE A-A-A A-B-B Af-Bf-B Af-Bf-B Af-Bf-B Af-Bf-Af 39B6-AYE A-A-AA-B-B Af-A-B Af-B-B B-B-B B-Af-Bf 39B6-ANK A-A-A A-A-B A-B-B B-A-B C-B-BD-Bf-Bf 39B6-ANR B-A-A B-A-B Af-Af-Bf Af-Af-Bf B-Af-Bf C-Af-Bf HighC-C-C C-C-C C-D-C D-D-C D-D-D D-D-D aggregation control PBSTw Af-B-BAf-Bf-B Af-Af-Bf Af-Bf-B Af-Bf-Bf Af-A-Af

For the 5° C. condition, the average scores for the mAbs after 56 dayswere as follows:

39B6-AYE: ‘sample is clear, no particles visible’

39B6-AVE and 39B6-ANK: ‘very few particles’

39B6-ANR: ‘moderate presence of particles’.

The PBSTw buffer is scored ‘sample is clear, no particles visible’ at 5°C. so this indicates that the observed particles are protein-related.

For the 37° C. storage condition, the average scores for the mAbs after56 days were as follows:

39B6-AYE, 39B6-ANK and 39B6-ANR: ‘very few particles’

39B6-AVE: ‘sample is clear, no particles visible’.

The PBSTw buffer is scored ‘sample is clear, no particles visible’ at37° C. so this indicates that the observed particles areprotein-related.

Analysis by SE-HPLC

Protein aggregation and fragmentation were measured by SE-HPLC.

The protein aggregation results for the mAbs 39B6-AVE, 39B6-AYE,39B6-ANK and 39B6-ANR, as measured by SE-HPLC, are summarized in Table20 below and shown in FIG. 5.

TABLE 20 % Aggregate formation monitored by SE-HPLC for mAbs 39B6-AVE,39B6-AYE, 39B6-ANK and 39B6-ANR 39B6-AVE - Percent aggregation (%) uponSE-HPLC analysis t (days) Ref 5° C. 37° C. 0 1.0 1.0 1.0 7 1.0 1.1 1.214 1.0 1.0 2.2 28 0.9 0.9 3.0 56 1.1 1.1 4.4 39B6-AYE - Percentaggregation (%) upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 0 1.21.0 1.0 7 1.0 1.0 1.0 14 1.0 1.0 1.1 28 0.9 0.9 1.4 56 1.1 1.0 0.539B6-ANK - Percent aggregation (%) upon SE-HPLC analysis t (days) Ref 5°C. 37° C. 0 1.0 1.0 1.0 28 0.9 0.9 1.1 56 0.9 0.9 0.7 39B6-ANR - Percentaggregation (%) upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 0 1.01.1 1.1 28 1.0 1.0 1.3 56 1.0 1.4 1.5

When stored at 5° C. no change in % aggregate levels was observed formAbs 39B6-AYE, 39B6-AVE and 39B6-ANK over the 56 d time period. Observed% aggregate levels were low—between 0.9% and 1.1%. A minor increase inaggregate levels was observed for mAb 39B6-ANR from 1.1% to 1.4%.

At 37° C., no change in % aggregate levels was observed for mAbs39B6-AYE and 39B6-ANK. For 39B6-ANR a minor increase from 1.1% at 0 d to1.5% at 56 d was observed. For 39B6-AVE an increase in % aggregate wasobserved from 1.0% at 0 d to 4.4% at 56 d.

The presence of fragment peaks for the mAbs 39B6-AVE, 39B6-AYE, 39B6-ANKand 39B6-ANR, as measured by SE-HPLC, is shown in Table 21 and FIG. 6.As fragmentation peaks were only observed at 37° C. after 56 days, onlythese results are presented.

TABLE 21 % Fragment formation monitored by size-exclusion chromatographyfor mAbs 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR at 56 days 39B6-AVE -Percent fragmentation (%) upon SE-HPLC analysis t (days) Ref 5° C. 37°C. 56 0 0 0.3 39B6-AYE - Percent fragmentation (%) upon SE-HPLC analysist (days) Ref 5° C. 37° C. 56 0 0 5.7 39B6-ANK - Percent fragmentation(%) upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 56 0 0 0.239B6-ANR - Percent fragmentation (%) upon SE-HPLC analysis t (days) Ref5° C. 37° C. 56 0 0 0.3

For mAbs 39B6-AVE, 39B6-ANK and 39B6-ANR the percentage of fragmentpeaks is between 0.2% and 0.3% after 56 days whilst for mAb 39B6-AYE thepercentage of fragment peaks is 5.7%.

The results of the % monomer peak for all mAbs are summarized in Table22.

TABLE 22 Monomer area (%) monitored by size-exclusion chromatography formAbs 39B6-AVE, 39B6-AYE, 39B6-ANK and 39B6-ANR 39B6-AVE - Percentmonomer area (%) upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 0 99.099.0 99.0 7 99.0 98.9 98.8 14 99.0 99.0 97.8 28 99.1 99.1 97.0 56 98.998.9 95.3 39B6-AYE - Percent monomer area (%) upon SE-HPLC analysis t(days) Ref 5° C. 37° C. 0 98.8 99.0 99.0 7 99.0 99.0 99.0 14 99.0 99.098.9 28 99.1 99.1 98.6 56 98.9 99.0 93.8 39B6-ANK - Percent monomer area(%) upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 0 99.0 99.0 99.0 2899.1 99.0 98.9 56 99.1 99.1 99.1 39B6-ANR - Percent monomer area (%)upon SE-HPLC analysis t (days) Ref 5° C. 37° C. 0 99.0 98.9 98.9 28 99.099.0 98.7 56 99.0 98.6 98.2SDS-PAGE

The SDS-PAGE results for analysis of ail antibodies are shown in Table23 and FIG. 8.

TABLE 23 Total percentage of full length Ab/heavy chain estimated bySDS-PAGE analysis and Odyssey scanning for mAbs 39B6-AVE, 39B6-AYE,39B6-ANK and 39B6-ANR Non-reducing conditions Reducing conditions t(days) Ref 5° C. 37° C. Ref 5° C. 37° C. % full-length 39B6-AVE  0 77.274.4 96.7 96.6  7 76.0 78.0 79.8 97.9 98.0 97.8 14 80.3 81.9 79.8 98.497.5 97.1 28 82.8 81.9 74.5 96.2 96.1 85.6 56 79.2 80.7 70.7 95.1 94.878.6 % full-length 39B6-AYE  0 74.2 74.6 96.4 97.2  7 76.5 79.6 77.898.4 97.8 98.0 14 77.1 77.9 76.9 97.1 96.5 90.8 28 75.6 77.3 73.6 97.098.3 90.3 56 73.4 73.4 59.8 96.4 94.7 88.0 % full-length 39B6-ANK  074.7 71.3 96.7 96.6 28 70.8 70.7 74.2 97.0 96.9 89.5 56 79.3 78.5 73.696.8 96.7 88.1 % full-length 39B6-ANR  0 76.2 76.5 97.1 96.9 28 78.178.7 73.5 96.9 96.1 84.9 56 74.1 75.7 70.6 95.5 95.6 79.0

No trend towards degradation was observed for any of the mAbs for the 5°C. temperature condition for both the reducing and non-reducingconditions.

At 37° C., all mAbs showed a similar rate of degradation for thenon-reducing condition except for mAb 39B6-AYE. This mAb shows a similardegradation rate up to 28 days but demonstrates a faster degradationrate between day 28 and day 56 compared to the other mAbs. For thereducing condition, all mAbs show a similar rate of degradation.

Target Binding Affinity on Biacore

Samples were tested for their target binding activity in Biacore bymeasuring the slope at each time point. The reference sample was set as100% of the activity. The results for all mAbs are summarized in Table24 and FIG. 9.

TABLE 24 Percentage activity on Biacore for mAbs 39B6-AVE, 39B6-AYE,39B6-ANK and 39B6-ANR 39B6-AVE - Activity on Biacore (%) t (days) Ref 5°C. 37° C. 0 100.0%  99.5%  99.5% 7 100.0% 111.7% 113.9% 14 100.0%  93.8% 90.2% 28 100.0%  99.0%  68.0% 56 100.0%  96.9%  31.3% 39B6-AYE -Activity on Biacore (%) t (days) Ref 5° C. 37° C. 0 100.0%  93.5%  93.5%7 100.0% 103.1% 101.5% 14 100.0%  97.5%  97.5% 28 100.0%  96.7%  96.1%56 100.0% 103.5% 104.2% 39B6-ANK - Activity on Biacore (%) t (days) Ref5° C. 37° C. 0 100.0% 130.1% 130.1% 28 100.0% 101.1%  77.8% 56 100.0%100.6%  62.4% 39B6-ANR - Activity on Biacore (%) t (days) Ref 5° C. 37°C. 0 100.0% 100.9% 100.9% 28 100.0% 129.1%  85.4% 56 100.0%  85.4% 46.3%

For the mAb samples stored at 37° C., only antibody 39B6-AYE retainedtarget binding activity over the full 56-day period. All of the otherantibodies displayed a significant decrease in target binding activityover the 56-day period when stored at 37° C.

Protein Concentration

The protein concentration of all samples was measured for each conditionon NanoDrop. In Table 25 and FIG. 10, the measured protein concentrationfor all mAbs is shown.

TABLE 25 Protein concentration (mg/ml) for mAbs 39B6-AVE, 39B6-AYE,39B6-ANK and 39B6-ANR 39B6-AVE - Protein concentration (mg/ml) t (days)Ref 5° C. 37° C. 0 1.1 1.0 1.0 7 1.0 1.0 1.1 14 1.0 1.0 1.1 28 1.0 1.01.0 56 1.0 1.0 1.1 39B6-AYE - Protein concentration (mg/ml) t (days) Ref5° C. 37° C. 0 1.0 1.0 1.0 7 1.0 1.0 1.0 14 1.0 1.0 1.0 28 1.0 1.0 1.056 1.0 1.0 1.0 39B6-ANK - Protein concentration (mg/ml) t (days) Ref 5°C. 37° C. 0 1.1 1.1 1.1 28 1.0 1.0 1.1 56 1.1 1.1 1.1 39B6-ANR - Proteinconcentration (mg/ml) t (days) Ref 5° C. 37° C. 0 1.0 1.0 1.0 28 1.0 1.01.0 56 1.0 1.0 1.0Summary and Conclusion for the Temperature Stability Study

The temperature stability study revealed some significant differencesbetween the four mAbs tested: an aggregation of 4.4% was observed onSE-HPLC for mAb 39B6-AVE after 56 days at 37° C. and a fragmentation of5.7% for mAb 39B6-AYE. However, this fragmentation for mAb 39B6-AYE didnot affect the target binding activity at 37° C. on Biacore; after 56days this was still as good as the reference sample. Meanwhile, lowertarget binding activity for mAbs 39B6-AVE, 39B6-ANK and 39B6-ANR wasclearly seen at 37° C. after 56 days.

2.2.2 Freeze-Thaw Stability Study

To monitor the stability of the antibodies under freeze-thaw conditions,the set-up was as follows. A 1 ml aliquot of the mAbs (at 5 mg/ml) wasfrozen for at least 6 hours at −20° C. and thawed for 1 hour at RT. Thiscycle was repeated 9× (10 freeze-thaw cycles in total). Samples wereanalyzed by visual inspection, SE-HPLC, SDS-PAGE, target bindingactivity on Biacore and protein concentration. Reference samples storedat −20° C. were used for all analyses in parallel. As the mAbs 39B6-ANRand 39B6-ANK have a possible deamidation site, they were only subjectedto analysis by visual inspection.

Visual Inspection

The results for visual inspection in the freeze-thaw stability study areshown in Table 26. All mAbs were in PBSTw, which should give astabilizing effect.

TABLE 26 Visual Inspection freeze-thaw stability for mAbs 39B6-AVE,39B6-AYE, 39B6-ANK and 39B6-ANR Sample 10x FT 39B6-AVE B-B-B 39B6-AYEB-Af-B 39B6-ANK A-Bf-Bf 39B6-ANR A-A-B High aggregation control C-D-DPBSTw B-Af-Bf

The average scores for the mAbs were as follows:

39B6-AVE, 39B6-AYE and 39B-ANK: ‘very few particles’; and

39B6-ANR: ‘sample is clear, no particles visible’.

The PBSTw buffer was scored by two people as ‘very few particles’ andtherefore, the particles seen in the 39B6-AVE, 39B6-AYE and 39B-ANKsamples may not be protein-related. It can be concluded that all mAbsremain unchanged after 10 freeze-thaw cycles.

Analysis by SE-HPLC

Protein aggregation and fragmentation were measured by SE-HPLC.

The protein aggregation results for the freeze-thaw stability study forthe mAbs 39B6-AVE and 39B6-AYE are summarized in Table 27.

TABLE 27 % Aggregate formation monitored by size-exclusionchromatography in freeze-thaw stability for mAbs 39B6-AVE and 39B6-AYE39B6-AVE 39B6-AYE Sample Ref 10x FT Ref 10x FT 1.0 1.0 1.0 0.9

No change in percentage aggregate levels was observed for either mAbfollowing 10 freeze-thaw cycles as compared to the reference samples.

The areas of the monomeric peak for all different injections were alsoexamined. The % monomer area results for the mAbs 39B6-AVE and 39B6-AYEare summarized in Table 28.

TABLE 28 % Monomer area monitored by size-exclusion chromatography infreeze-thaw stability for mAbs 39B6-AVE and 39B6-AYE. 39B6-AVE 39B6-AYESample Ref 10x FT Ref 10x FT 99.0 99.0 99.0 99.1

No change in % monomer area was observed for both mAbs following 10freeze-thaw cycles compared to the reference samples.

Analysis by SDS-PAGE

Freeze-thaw samples were analyzed for their integrity by SDS-PAGE undernon-reducing and reducing conditions. The results are shown in Table 29and FIG. 11 for mAbs 39B6-AVE and 39B6-AYE.

TABLE 29 Total percentage of full length Ab/heavy chain estimated bySDS-PAGE analysis and Odyssey scanning in freeze-thaw stability studyfor mAbs 39B6-AVE and 39B6-AYE % full-length 39B6-AVE Non-reducingconditions Reducing conditions Sample Ref 10x FT Ref 10x FT 78.1 79.395.9 96.1 % full-length 39B6-AYE Non-reducing conditions Reducingconditions Sample Ref 10x FT Ref 10x FT 78.8 78.7 96.3 97.0

No changes were observed for both mAbs after 10 freeze-thaw cycles.

Analysis of Target Binding by Biacore

Samples were tested for their target binding activity in Biacore bymeasuring the slope after 10 freeze-thaw cycles. The reference samplewas set as 100% of the activity. The results for both mAbs 39B6-AVE and39B6-AYE are shown in FIG. 12.

The results demonstrate that after 10 freeze-thaw cycles no change intarget binding activity is observed.

Analysis of Protein Concentration

The protein concentration of the freeze-thaw samples was measured onNanoDrop.

In Table 30, the measured protein concentration for mAbs 39B6-AVE and39B6-AYE after 10 freeze-thaw cycles is shown. Also, FIG. 13 shows theseresults for both mAbs.

TABLE 30 Protein concentration (mg/ml) in freeze-thaw stability for mAbs39B6-AVE and 39B6-AYE 39B6-AVE 39B6-AYE Sample Ref 10x FT Ref 10x FT 1.01.1 1.0 1.0Conclusion

The freeze-thaw stability study did not reveal any significantdifferences for the tested mAbs 39B6-AVE and 39B6-AYE.

2.2.3 Thermal Stability Study

To analyse the melting curves, mAbs were heated according to the schemebelow. Following completion of the thermal cycle in the PCR device,samples were tested for affinity on Biacore.

The protocol used was as follows:

-   -   1) Aliquots of 1 ml for each mAb were stored in glass vials at        −20° C. in the beginning of the study    -   2) After one week of storage, they were defrosted once    -   3) The mAbs were diluted at 1 mg/ml as usual and then further        diluted at 100 μg/ml (10× dilution: 200 μl+1800 μl PBSTw)    -   4) The diluted mAbs were aliquoted in a PCR plate (50 μl/well)    -   5) Keep sufficient sample and also sample at 5° C. as reference        to be analyzed in parallel    -   6) 1 h in PCR device exposed at the temperatures given below    -   7) 2 h in PCR device at 25° C.    -   8) At 4° C. in PCR device    -   9) Prepare the samples and the references for analysis at 4        μg/ml (25× dilution: 168 μl Biacore buffer+7 μl sample)

Run the program as shown in FIG. 21 in a gradient PCR device.

Protocol for Biacore Analysis:

-   -   The same CM5 chip, coated with ˜4000 RU human GARP-TGF-β complex        was used    -   Open the curves in the BIAEvaluation program and select value        ‘2-1’ (1: blank, 2 hGARP-TGF-β)    -   Select one curve at the time for plot overlay    -   Delete the regeneration part of the curve    -   Select the baseline just before the injection and transform the        Y-axis for ‘zero at median of selection’    -   Select ‘General Fit’, starting from 120 sec after injecting and        ending at 155 sec    -   For calculation of the IC50: The slope for 5° C. references is        leveled at 100%. The percentage (Y-axis) of the other        temperatures (X-axis) can be calculated

Thermo-tolerance of the four mAbs was measured and the results aresummarized in FIG. 14. The melting temperature was calculated as thetemperature at which 50% of the antibody is still functional. Thereference samples which were kept at 5° C. were set as 100% of theactivity. The melting temperatures are shown in Table 31.

TABLE 31 Melting temperatures in thermal stability study mAb Meltingtemperature 39B6-AVE 67.0° C. 39B6-AYE 66.5° C. 39B6-ANK 69.1° C.39B6-ANR 68.6° C.

The mAbs 39B6-ANK and 39B6-ANR displayed the highest meltingtemperatures: 69.13° C. and 68.55° C., respectively. The mAbs 39B6-AVEand 39B6-AYE also gave good melting temperatures, 66.99° C. and 66.53°C., respectively. All melting temperatures for all mAbs can beconsidered high.

Conclusion of the Thermal Stability Study

The four mAbs demonstrated good thermo-tolerance. The meltingtemperatures were comparable to the original 39B6-A mAb in the previousstability study, 66.8° C.

2.2.4 Rotational Stability Study

For the rotational stability study, the set-up was as follows. Aliquotsof 1 ml for each mAb were stored in glass vials at −20° C. at thebeginning of the study. After one week of storage the aliquots weredefrosted once and rotated head over head at 15 rpm at room temperature.

Samples were scored for presence of particles at the indicated timepoints:

-   -   Hours: 0, 3, 6, 24, 30, 48, 54, 72 and 96

Samples were also analyzed after 96 hours by SE-HPLC, SDS-PAGE, targetbinding activity on Biacore and protein concentration. Reference samplesstored at −20° C. were used for all analyses in parallel. As the mAbs39B6-ANR and 39B6-ANK still have a possible deamidation site, they wereonly assessed by visual inspection.

Visual Inspection

The results for visual inspection in the rotational stability study areshown in Table 32. All mAbs are in PBSTw, which should give astabilizing effect.

TABLE 32 Visual Inspection rotational stability study for mAbs 39B6-AVE,39B6-AYE, 39B6-ANK and 39B6-ANR Sample 0 h 3 h 6 h 24 h 30 h 48 h 54 h72 h 96 h 39B6-AVE Af-B-Bf Af-B-Bf Af-B-Bf Af-Cf-Cf Af-Bf- Af-B- B-B-B-B- B-B- Cf Bf Bf Bf Bf 39B6-AYE Af-Bf-Bf Af-Bf-Bf Af-Bf-Bf Af-Bf-BfAf-Bf- Af-Bf- Af-Bf- B-Bf- B-Bf- Bf Bf Bf Bf Bf 39B6-ANK Af-Bf-CAf-Bf-Bf Af-Bf-Bf Af-Bf-Bf Af-Bf- Af-Bf- B-Bf- B-Bf- B-Bf- Bf Bf Cf CfCf 39B6-ANR Af-Bf-Bf Af-Bf-Bf Af-Bf-Bf Af-Bf-Bf Af-Bf- Af-Bf- Af-Bf-Af-Bf- Af-Bf- Bf Bf Bf Bf Bf High C-B-C C-Df-D C-Df-D C-Df-Df C-Df-C-Df- C-Df- D-Df- D-Df- aggregation Df Df Df Df Df control PBSTw A-A-AA-A-A A-A-A A-A-A A-A-A A-A-A A-A-A A-A-A A-A-A

All mAbs were found to have an average score of ‘very few particles’ sothey remain relatively unaffected after 96 hours of rotation. The PBSTwbuffer did not contain any particles in any experimental conditiontested. This indicates that the observed ‘very few particles’ areprotein-related in the antibody samples.

Analysis by SE-HPLC

Protein aggregation and fragmentation were measured by SE-HPLC. Theprotein aggregation results for the rotational stability study for themAbs 39B6-AVE and 39B6-AYE are shown in Table 33.

TABLE 33 % Aggregate formation monitored by size-exclusionchromatography in rotational stability study for mAbs 39B6-AVE and39B6-AYE. 39B6-AVE 39B6-AYE Sample Ref 96 h Ref 96 h 1.1 1.0 1.0 1.0

No change in % aggregate levels were observed for the mAbs followingrotational stress as compared to the reference samples.

The areas of the monomeric peak for all different injections were alsoexamined. The % monomer area results for the mAbs 39B6-AVE and 39B6-AYEare shown in Table 34.

TABLE 34 % Monomer area monitored by size-exclusion chromatography inrotational stability study for mAbs 39B6-AVE and 39B6-AYE 39B6-AVE39B6-AYE Sample Ref 96 h Ref 96 h 98.9 99.0 99.0 99.0

No change in % monomer area was observed for either mAb after 96 hoursof rotation as compared to the reference samples.

For both antibodies, no change in SE-HPLC profile was observed after 96hours of rotation as compared to the reference samples.

Analysis by SDS-PAGE

Rotation samples were analyzed for their integrity by SDS-PAGE undernon-reducing and reducing conditions. The results are summarized inTable 35 for mAbs 39B6-AVE and 39B6-AYE.

TABLE 35 Total percentage of full length Ab/heavy chain estimated bySDS-PAGE analysis and Odyssey scanning in rotational stability study formAbs 39B6-AVE and 39B6-AYE % full-length 39B6-AVE Non-reducingconditions Reducing conditions Ref 96 hours Ref 96 hours Sample 81.376.6 91.0 90.7 % full-length 39B6-AYE Non-reducing conditions Reducingconditions Sample Ref 96 hours Ref 96 hours 76.2 79.0 91.5 92.6

The SDS-PAGE gel for both mAbs after 96 hours of rotation can be seen inFIG. 15.

No changes were observed for either antibody after 96 hours of rotation.

Analysis for Target Binding in Biacore

Samples were tested for their target binding activity in Biacore bymeasuring the slope after 96 hours of rotation. The reference sample wasset as 100% of the activity. The results for both mAbs 39B6-AVE and39B6-AYE are shown in FIG. 16.

Analysis for Protein Concentration

The protein concentration of the rotational samples was measured onNanoDrop.

In Table 36, the measured protein concentration for mAbs 39B6-AVE and39B6-AYE after 96 hours of rotation is shown. Also, FIG. 17 shows theseresults for both mAbs.

TABLE 36 Protein concentration (mg/ml) in rotational stability for mAbs39B6-AVE and 39B6-AYE 39B6-AVE 39B6-AYE Sample Ref 96 h Ref 96 h 1.0 1.11.0 1.1

No protein loss was observed for both mAbs after 96 hours of rotation.

Conclusion of the Rotational Stability Study

The rotational stability study did not reveal any difference between thetested mAbs 39B6-AVE and 39B6-AYE.

2.2.5 Primary Sequence Analysis by Peptide Mapping

Samples from the temperature, freeze-thaw and rotational stability studywere analyzed using Tryptic peptide mapping RP-HPLC-UV-MS methodology toidentify modifications (deamidation, isomerization and oxidation) at theprotein (peptide) level.

Deamidation in the CDR3 of the heavy chain at the positions 95-96 (aminoacids NE) was engineered out in mAbs 39B6-AVE and 39B6-AYE by mutationof position N95. This deamidation site is still present in mAbs 39B6-ANKand 39B6-ANR and significant deamidation/isomerization was detectedafter 28 days at both 5° C. and 37° C. in the temperature stabilitystudy and also in the freeze-thaw and rotational stability study. It wasconcluded that deamidation could not be prevented by the introduction ofbulky positively charged residues downstream of N95.

At position 100f of the CDR3 of the heavy chain there is a methioninethat is essential for high binding affinity. Temperature, freeze-thawand rotational stability studies have demonstrated that oxidation ofM100f does not occur in mAbs 39B6-AYE and 39B6-ANK, while oxidation isstill observed for mAbs 39B6-AVE and 39B6-ANR. This demonstrates thatthe amino acids at positions 95 and 96 influence the sensitivity towardsoxidation of the downstream methionine. Table 37 shows an overview ofthe levels of oxidation and deamidation of peptides covering the heavychain CDR3, which were generated by tryptic digestion.

TABLE 37 Extent of oxidation and deamidation of peptides within theheavy chain CDR3 Oxidation (%) Deamidation (%) 5° C. 37° C. Freeze 5° C.37° C. Freeze day day Rotational thaw day day Rotational thaw 28 28 (96h) (10×) 28 28 (96 h) (10×) 39B6-AVE 1.08 10.51 1.58 3.47 0 0 0 039B6-AYE Below the limit of quantitation (LOQ) 0 0 0 0 39B6- Below thelimit of quantitation (LOQ) 4.72 11.15 3.5 4.75 ANK 39B6- 9.20 20.9819.37 12.47 12.85 34.38 12.79 12.77 ANR *Below the limit of quantitation(LOQ) = intensity of oxidized form is close to background

The relative amount of deamidation and isomerization of N95, and therelative binding activity of the variants stored at 37° C. are depictedin FIG. 18. Also the original 39B6-A is displayed in the figure(labelled 39B6-ANE). Because N95 has been mutated in mAbs 39B6-AVE and39B6-AYE, deamidation and isomerization levels are zero and thereforenot included. The correlation between deamidation of N95 and lowertarget binding activity observed for mAbs 39B6-ANK and 39B6-ANR suggeststhat N95 deamidation negatively affects the binding of the mAb to itstarget.

Conclusion

The GARP-TGF-β1 antibody variant 39B6-AYE is a particularly goodGARP-TGF-β1 antibody to take forward for clinical development becauseit:

-   -   Retains high affinity binding to its target, the GARP-TGF-β1        complex;    -   Displays good potency in the SMAD2 phosphorylation assay;    -   Does not undergo deamidation or isomerization in CDR3 Does not        undergo oxidation in CDR3    -   Displays high human homology (95%)    -   Displays improved stability as compared with 39B6-A, as measured        by different stability assays.

The CDR and variable domain sequences for 39B6-AYE are shown in Tables38 and 39 below. The full-length heavy chain and light chain sequencesare shown in Table 40. The polynucleotide sequences encoding the VH andVL domains and the full-length heavy and light chains are shown in Table41.

TABLE 38 VH and VL CDR sequences for 39B6-AYE 39B6-AYE SEQ ID NO: VHCDR1 SYYID  4 CDR2 RIDPEDAGTKYAQKFQG 12 CDR3 YEWETVVVGDLMYEYEY 13 VLCDR1 QASQSISSYLA  9 CDR2 GASRLKT 10 CDR3 QQYASVPVT 11

TABLE 39 VH and VL domain sequences for 39B6-AYE SEQ ID 39B6-AYE NO: VHQVQLVQPGAEVRKPGASVKVSCKASGYRFTSYY 14 IDWVRQAPGQGLEWMGRIDPEDAGTKYAQKFQGRVTMTADTSTSTVYVELSSLRSEDTAVYYCARY EWETVVVGDLMYEYEYWGQGTLVTVSS VLDIQMTQSPSSLSASVGDRVTITCQASQSISSYL 15 AWYQQKPGQAPKILIYGASRLKTGVPSRFSGSGSGTSFTLTISSLEPEDAATYYCQQYASVPVTFG QGTKVEIK

TABLE 40 Heavy chain and light chain sequences for 39B6-AYE SEQ 39B6-AYEID NO: Heavy QVQLVQPGAEVRKPGASVKVSCKASGYRFTSYY 16 chainIDWVRQAPGQGLEWMGRIDPEDAGTKYAQKFQG RVTMTADTSTSTVYVELSSLRSEDTAVYYCARYEWETVVVGDLMYEYEYWGQGTLVTVSSASTKGP SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Light DIQMTQSPSSLSASVGDRVTITCQASQSISSYL 17 chainAWYQQKPGQAPKILIYGASRLKTGVPSRFSGSG SGTSFTLTISSLEPEDAATYYCQQYASVPVTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

TABLE 41 Polynucleotide sequences encoding 39B6-AYE SEQ ID NO: VHCAAGTCCAACTTGTCCAACCGGGGGCGGAAGTGCG 18GAAGCCGGGGGCGAGCGTGAAAGTCTCGTGCAAGGCATCGGGATACCGATTCACATCATATTACATCGACTG GGTCAGGCAAGCGCCGGGGCAAGGGCTGGAATGGATGGGGCGGATCGACCCGGAGGATGCCGGGACGAA ATATGCGCAAAAATTCCAAGGGCGCGTCACGATGACGGCCGACACATCGACGAGCACGGTATACGTGGAGC TGAGCTCGCTGAGGAGCGAGGACACCGCGGTATACTACTGCGCGCGATACGAATGGGAGACCGTCGTCGT CGGGGACCTGATGTACGAATACGAATACTGGGGGCAAGGGACGCTTGTCACGGTCTCGAGC VL GACATCCAGATGACTCAGAGCCCTTCCAGCCTGAGC 19GCCTCTGTGGGAGATAGAGTCACCATCACATGCCAGGCTAGTCAGTCAATTTCTAGTTACCTGGCATGGTATCAGCAGAAGCCTGGCCAGGCACCTAAAATCCTGATCT ACGGAGCCAGTAGGCTGAAGACAGGGGTGCCATCTCGGTTCTCCGGCAGCGGATCTGGGACATCCTTTACTCTGACCATCTCATCCCTGGAGCCAGAAGACGCCGCTACATACTATTGTCAGCAGTATGCTTCCGTGCCCGTC ACATTCGGTCAGGGCACTAAGGTCGAGATCAAGHeavy CAAGTCCAACTTGTCCAACCGGGGGCGGAAGTGCG 20 chainGAAGCCGGGGGCGAGCGTGAAAGTCTCGTGCAAGGCATCGGGATACCGATTCACATCATATTACATCGACTG GGTCAGGCAAGCGCCGGGGCAAGGGCTGGAATGGATGGGGCGGATCGACCCGGAGGATGCCGGGACGAA ATATGCGCAAAAATTCCAAGGGCGCGTCACGATGACGGCCGACACATCGACGAGCACGGTATACGTGGAGC TGAGCTCGCTGAGGAGCGAGGACACCGCGGTATACTACTGCGCGCGATACGAATGGGAGACCGTCGTCGT CGGGGACCTGATGTACGAATACGAATACTGGGGGCAAGGGACGCTTGTCACGGTCTCGAGCGCTAGCACC AAGGGCCCCTCCGTGTTCCCCCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGGCT GCCTGGTGAAAGACTACTTCCCCGAGCCTGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGCGTG CACACCTTCCCTGCCGTGCTGCAATCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCCTCCTCCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGAC CACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAATCTAAATACGGCCCTCCCTGCCCCCCCTGCCCTGCCCCTGAATTTCTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTC CCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCAGAGGTGCAGTTCAACTGGT ATGTTGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTACCGG GTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCAGCATCGAAAAGACCATCTC CAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAGGAAGAGATGACCAAG AACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTGGAATGGGAGTCCAAC GGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTC TCGGCTGACAGTGGATAAGTCCCGGTGGCAGGAAGGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTATACCCAGAAGTCCCTGTCCCTGAGCCTGGGC Light GACATCCAGATGACTCAGAGCCCTTCCAGCCTGAGC 21 chainGCCTCTGTGGGAGATAGAGTCACCATCACATGCCAGGCTAGTCAGTCAATTTCTAGTTACCTGGCATGGTATCAGCAGAAGCCTGGCCAGGCACCTAAAATCCTGATCT ACGGAGCCAGTAGGCTGAAGACAGGGGTGCCATCTCGGTTCTCCGGCAGCGGATCTGGGACATCCTTTACTCTGACCATCTCATCCCTGGAGCCAGAAGACGCCGCTACATACTATTGTCAGCAGTATGCTTCCGTGCCCGTCACATTCGGTCAGGGCACTAAGGTCGAGATCAAGCGTACGGTCGCGGCGCCTTCTGTGTTCATTTTCCCCCCATCTGATGAACAGCTGAAATCTGGCACTGCTTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCCAAAGTCCAGTGGAAAGTGGACAATGCTCTGCAGAGTGGGAATTCCCAGGAATCTGTCACTGAGCAGGACTCTAAGGATAGCACATACTCCCTGTCCTCTACTCTGACACTGAGCAAGGCTGATTACGAGAAACACAAAGTGTACGCCTGTGAAGTCACACATCAGGGGCTGTCTAGTCCT GTGACCAAATCCTTCAATAGGGGAGAGTGC

Example 3. Batch Testing of 39B6-AYE (ARGX-115)

The pilot drug substance batch of ARGX-115 was tested for stability overa three-month period. Test samples of the pilot drug substance batchwere stored at the intended storage condition of −70° C., at theaccelerated storage condition of +5° C. and the stressed storagecondition of +25° C. The pilot drug substance was presented in theformulation: 10 mM Histidine/Histidine Hydrochloride, 200 mM Sucrose, 40mM Arginine, 0.03% (w/v) polysorbate 80 at pH 6.0 and a proteinconcentration of 20.0±2.0 mg/ml.

ARGX-115 pilot drug substance batch was confirmed to be stable for threemonths when stored at the intended storage condition of −70° C. The SPRbinding activity was also determined as a measure of the stability. Thebinding activity was expressed as a percentage of the binding activityof the reference sample that was kept at −70° C.

The results are shown in Table 42 below.

TABLE 42 Method Samples Results ARGX115 Biacore T3M +5° C. 101% T3M −70°C. 106% T3M +25° C. 107%

These results confirm that ARGX-115 is stable over a prolonged storageperiod.

Example 4. Characterisation of ARGX-115 Binding to the GARP-TGF-βComplex

4.1 Mature TGF-β is Essential for ARGX-115 Binding

In nature, the GARP-TGF-β complex (GARP in complex with latent TGF-β) isformed in the endoplasmic reticulum with covalent cysteine interactions(disulphide bridges) between GARP and latent TGFβ. This complex is thendisplayed on the cell surface. In vitro, the GARP-TGF-β complex can beformed from recombinant human GARP and recombinant human latent TGF-β(C33S)-3× strep-tag. The C33S mutant form of latent TGF-β does not formthe covalent interactions with GARP (or any of the Latent TGF-β BindingProteins (LTBPs)) like are present in the native complex.

To demonstrate ARGX-115 binding to the in vitro-formed complex ofrecombinant GARP and recombinant latent TGFβ (C33S), recombinant GARPwas coated to an ELISA plate (1 μg/mL human GARP 0/N at 4° C.), blockedwith blocking agent (casein-PBS), and latent TGF-β (5 μg/mL 1 h RT) wascaptured by the coated GARP. ARGX-115 and an isotype control antibody (1μg/mL 1 h at RT) were allowed to bind to the complex, and were detectedwith a HRP-conjugated anti-human IgG. As shown in FIG. 19, ARGX-115bound to the GARP-latent TGF-β (C33S) complex, whereas the isotypecontrol did not.

The assay was also found to work the other way around. The ELISA platewas coated with ARGX-115, or an isotype control antibody (1 μg/mL ON at4° C.) and blocked with blocking agent (casein-PBS). Recombinant GARP (5μg/mL) was captured by the coated ARGX-115 antibody, the plate waswashed, and recombinant latent TGF-β (C33S)-3× strep-tag (5 μg/mL) wascaptured and detected with streptavidin-HRP. HRP activity was detectedonly in the presence of ARGX-115 and not in wells of the plate coatedwith the isotype control.

To test the binding of ARGX-115 to a complex of GARP and the latencyassociated peptide (LAP) of TGF-β, a complex between recombinant GARPand recombinant LAP was formed. An ELISA plate was coated withrecombinant GARP (1 μg/mL O/N 4° C.), blocked with blocking agent(casein-PBS), and LAP (5 μg/mL) was captured on the coated recombinantGARP. LAP binding was detected with anti-LAP-HRP. The binding of theanti-LAP-HRP demonstrates that the GARP-LAP complex does form in vitro.ARGX-115, however, did not show any binding to the GARP-LAP complex.Moreover, when the ELISA plate was coated with ARGX-115 (1 μg/mL ON 4°C.), recombinant GARP (5 μg/mL) was added followed by LAP (5 μg/mL), nobinding of anti-LAP-HRP was measured. These results confirm that matureTGF-β is required for ARGX-115 binding to the GARP-TGF-β complex.

4.2 Impact of Mutations in hTGF-β in Complex with GARP on theNeutralizing Activity of ARGX-115

293T cells stably expressing integrin αvβ6 (293Tcl.ITGB6) weretransiently transfected with a mix of 3 plasmids (plasmid mix): (1)CAGA-luc reporter plasmid; (ii) human GARP (pEF-BOS-puro-hGARP); and(iii) either WT human TGF-β or mutant TGF-β (pDisptay). Integrin αvβ6 isone of the two TGF-β-activation integrins. 293Tcl.ITGB6 cells weredetached and harvested from semi-confluent 75 cm² flasks, counted anddiluted to 1E+06 cells/mL, and distributed 1 mL per eppendorf tube. 250μl plasmid mix was added to each tube for transfection. Directly aftertransfection, the transfected cells were distributed in a 96-welloptical plate, at 50 μl (4E+04 cells) per well, containing differenttest mAbs (ARGX-115, LHG10.6, 1D11 and MHG-8 at 100 μl/mL). 1D11 is amAb against the active from of TGF-β isoform-1, -2 and -3. MHG-8 andLHG10.6 are described in WO2015/015003 and WO2016/125017. Afterincubation for 24 h at 37° C., the luciferase activity was measured. Thevalue obtained with the transfection of mutant TGF-β was expressed as apercentage of the value obtained for WT-TGF-β, The results are shown inFIG. 20. As can be seen from the figure, the neutralizing activity ofARGX-115 measured against the TGF-β mutant including the R58Asubstitution and the TGF-β mutant including the K338E substitution wassignificantly reduced. This indicates that these two residues in theGARP-TGF-β complex are particularly important for the neutralizingactivity of ARGX-115.

The invention claimed is:
 1. A recombinant antibody or antigen bindingfragment thereof, which binds to a complex of human glycoprotein Arepetitions predominant (GARP) and TGF-β1, wherein the antibody orantigen binding fragment thereof comprises a heavy chain variable domain(VH), wherein: the VH CDR3 comprises the amino acid sequenceYEWETVVVGDLMYEYEY (SEQ ID NO: 13), the VH CDR2 comprises the amino acidsequence RIDPEDAGTKYAQKFQG (SEQ ID NO: 12), and the VH CDR1 comprisesthe amino acid sequence SYYID (SEQ ID NO: 4); and a light chain variabledomain (VL), wherein: the VL CDR3 comprises the amino acid sequenceQQYASVPVT (SEQ ID NO: 11), the VL CDR2 comprises the amino acid sequenceGASRLKT (SEQ ID NO: 10), and the VL CDR1 comprises the amino acidsequence QASQSISSYLA (SEQ ID NO: 9).
 2. The antibody or antigen bindingfragment of claim 1, wherein the heavy chain variable domain (VH)comprises the amino acid sequence of SEQ ID NO: 14, and the light chainvariable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15.3. The antibody or antigen binding fragment of claim 1, furthercomprising the CH1 domain, hinge region, CH2 domain and/or CH3 domain ofa human IgG.
 4. The antibody or antigen binding fragment of claim 3,wherein the human IgG is IgG1.
 5. The antibody or antigen bindingfragment of claim 3, wherein the human IgG is IgG4.
 6. The antibody orantigen binding fragment of claim 5, wherein the human IgG4 has thesubstitution S228P in the CH3 domain.
 7. An isolated polynucleotidewhich encodes the heavy chain variable domain of the antibody or antigenbinding fragment of claim
 1. 8. The isolated polynucleotide of claim 7,which comprises the sequence of SEQ ID NO:18.
 9. An isolatedpolynucleotide which encodes the light chain variable domain of theantibody or antigen binding fragment of claim
 1. 10. The isolatedpolynucleotide of claim 9, which comprises the sequence of SEQ ID NO:19.11. An isolated polynucleotide which encodes the antibody or antigenbinding fragment of claim
 1. 12. An expression vector comprising thepolynucleotide of claim 11 operably linked to regulatory sequences whichpermit expression of the antibody, antigen binding fragment, heavy chainvariable domain, or light chain variable domain in a host cell orcell-free expression system.
 13. A host cell or cell-free expressionsystem containing the expression vector of claim
 12. 14. A method ofproducing a recombinant antibody or antigen binding fragment thereof,comprising culturing the host cell or cell-free expression system ofclaim 13 under conditions which permit expression of the antibody orantigen binding fragment; and recovering the expressed antibody orantigen binding fragment.
 15. A pharmaceutical composition comprisingthe antibody or antigen binding fragment according to claim 1 and atleast one pharmaceutically acceptable carrier or excipient.
 16. Arecombinant antibody, which binds to a complex of human glycoprotein Arepetitions predominant (GARP) and TGF-β1, wherein the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:16, and a light chain comprising the amino acid sequence of SEQ ID NO:17.